RU2782358C2 - Cas-expressing mouse embryonic stem cells, and mice and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a mouse containing an expression cassette Cas9 genomically integrated into locus Rosa26, wherein the expression cassette Cas9 contains an encoding sequence for protein Cas9, to a cell of the specified mouse, as well as to its production method.

EFFECT: invention is effective for testing a capability of CRISPR/Cas9-nuclease containing protein Cas9 and guiding RNA of modifying a target genomic locus in vivo, as well as for optimization of the capability of CRISPR/Cas9-nuclease of modifying the target genomic locus in vivo.

67 cl, 27 dwg, 12 tbl, 2 ex

Description

Link to related applications

[0001] This application claims priority under U.S. Patent Application No. 62/539,275, filed July 31, 2017, which is incorporated herein by reference in its entirety for all purposes.

Link to a sequence listing provided as a text file via the EFS file system

[0002] The sequence listing provided in file 516569SEQLIST.txt is 178 KB in size and was created on July 30, 2018 and incorporated herein by reference.

Background of the Invention

[0003] CRISPR/Cas technology is a promising new therapeutic tool. However, there is a need for more efficient means of evaluating the efficiency of generating mutations or targeted modification of genes by an administered CRISPR/Cas agent in vivo. One of the limitations of testing the system in vivo is the need for simultaneous introduction of all components into a living organism. A typical way to introduce these components is to transiently transfect the DNA constructs into cells that will generate the appropriate RNA and protein. While this approach is effective, it has an inherent disadvantage as cells must rely on plasmid DNA constructs to first undergo transcription and then translation before the Cas9 protein is available to interact with the sgRNA component. Better methods and tools are needed to better evaluate the activity of administered CRISPR/Cas agents and to evaluate different delivery methods and parameters to target specific tissues or cell types in vivo.

[0004] In addition, the delivery of biologically active agents, such as CRISPR/Cas agents, to subjects is often hampered by difficulties in reaching the target cell or tissue components. These limitations may lead, for example, to the need to use much higher concentrations of agents than is desirable to achieve the result, which increases the risk of toxic effects and side effects. Improved delivery methods and methods for evaluating such delivery methods in vivo are needed.

Brief summary of the present invention

[0005] Cas9-ready, hereinafter referred to as Cas9-expressing (i.e., ready-to-use Cas-based genome-editing systems) non-human animals are provided, and methods and compositions are provided for evaluating the ability of CRISPR/Cas-nuclease agents to modify target genomic locus in vivo. In one aspect, methods are provided for testing the ability of a CRISPR/Cas nuclease to modify a target genomic locus in vivo. Some such methods include: (a) administering to a non-human animal a guide RNA designed to target a target sequence for the guide RNA at a target genomic locus, wherein the non-human animal comprises a genomically integrated Cas expression cassette containing the NLS-Cas coding sequence, and wherein the guide RNA is introduced via adeno-associated virus (AAV) mediated delivery; and (b) assessing the modification of the target genomic locus. Some such methods include: (a) administering to a non-human animal a guide RNA designed to target a target sequence for the guide RNA at a target genomic locus, wherein the non-human animal comprises a genomically integrated Cas expression cassette containing the NLS-Cas coding sequence, and wherein the guide RNA is introduced via lipid nanoparticle (LNP) mediated delivery; and (b) assessing the modification of the target genomic locus.

[0006] According to some such methods, the AAV is AAV7, AAV8 or AAV9, and step (b) involves assessing the modification of the target genomic locus in the liver. Optionally, AAV is AAV8.

[0007] According to some such methods, the route of administration of AAV to a non-human animal is intravenous injection, intraparenchymal injection, intraperitoneal injection, nasal instillation, or intravitreal injection.

[0008] According to some such methods, an exogenous donor nucleic acid is introduced in step (a), wherein the exogenous donor nucleic acid is intended to recombine with a target genomic locus. Optionally, the exogenous donor nucleic acid is a single stranded oligodeoxynucleotide (ssODN).

[0009] According to some such methods, the non-human animal is a rat or mouse. Optionally, the non-human animal is a mouse.

[0010] According to some such methods, the target genomic locus contains the target gene, and step (b) involves measuring the expression of the target gene or the activity of the protein encoded by the target gene.

[0011] In some such methods, step (b) involves sequencing the target genomic locus in one or more cells isolated from a non-human animal.

[0012] According to some such methods, step (b) involves isolating a target organ or tissue from a non-human animal and assessing the modification of the target genomic locus in the target organ or tissue. Optionally, step (b) involves assessing the modification of the target genomic locus in two or more different cell types within the target organ or tissue.

[0013] According to some such methods, step (b) involves isolating a non-target organ or tissue from a non-human animal and assessing the modification of the target genomic locus in the non-target organ or tissue.

[0014] According to some such methods, the NLS-Cas coding sequence is an NLS-Cas9 coding sequence.

[0015] According to some such methods, the Cas expression cassette further comprises a polyadenylation signal located upstream of the NLS-Cas coding sequence, wherein the polyadenylation signal is flanked by recombinase recognition sites, and wherein the polyadenylation signal in the Cas expression cassette has been excised in a tissue-specific manner. Optionally, the polyadenylation signal located upstream of the NLS-coding sequence in the Cas expression cassette was excised in the liver. Optionally, the recombinase that recognizes the recombinase recognition sites in the Cas expression cassette is a Cre recombinase. Optionally, the non-human animal further comprises a genomically integrated Cre recombinase expression cassette, wherein the Cre recombinase expression cassette contains a Cre recombinase coding sequence operably linked to a tissue-specific promoter. Optionally, the Cre recombinase gene is operably linked to one of the promoters shown in Table 2.

[0016] In some such methods, the Cas expression cassette further comprises a polyadenylation signal upstream of the NLS-Cas coding sequence, wherein the polyadenylation signal is flanked by recombinase recognition sites, and the method further comprises administering the recombinase to the non-human animal in a tissue-specific manner. Optionally, the recombinase is administered via adeno-associated virus (AAV) mediated delivery or lipid nanoparticle (LNP) mediated delivery. Optionally, the recombinase is administered via AAU8-mediated delivery. Optionally, the recombinase is administered to the liver.

[0017] In some such methods, the Cas expression cassette further comprises a fluorescent protein coding sequence. Optionally, the Cas expression cassette comprises a multicistronic nucleic acid comprising an NLS-Cas coding sequence and a fluorescent protein coding sequence separated by an internal ribosome entry intermediate site (IRES) or an intermediate 2A peptide coding sequence. Optionally, the multicistronic nucleic acid in the Cas expression cassette contains an NLS-Cas coding sequence and a green fluorescent protein coding sequence separated by an intermediate P2A peptide coding sequence. In some such methods, the Cas expression cassette additionally does not contain a fluorescent protein coding sequence. In some such methods, the NLS-Cas coding sequence encodes a Cas protein containing a protein tag.

[0018] In some such methods, the Cas expression cassette is operably linked to an endogenous promoter. In some such methods, the Cas expression cassette is operably linked to an exogenous, constitutive promoter.

[0019] In some such methods, the 5' end of the Cas expression cassette further comprises a 3' splicing sequence.

[0020] According to some such methods, the Cas expression cassette encodes a protein containing the sequence shown in SEQ ID NO: 13, 16, 19, or 22. Optionally, the Cas expression cassette contains the sequence shown in SEQ ID NO: 28, 29, 30, or 31 Optionally, the Cas expression cassette contains the sequence shown in SEQ ID NOs: 1, 12, 14, 15, 17, 18, 20, or 21.

[0021] In some such methods, the Cas expression cassette is integrated into a safe harbor locus. Optionally, the safe harbor locus is the Rosa26 locus. Optionally, the Cas expression cassette is integrated into the first intron of the Rosa26 locus.

[0022] According to some such methods, the non-human animal is heterozygous for the Cas expression cassette. In some such methods, the non-human animal is homozygous for the Cas expression cassette.

[0023] In some such methods, the non-human animal is a mouse, the AAV is AAV8, the Cas expression cassette is operably linked to the endogenous Rosa26 promoter, is inserted into the first intron of the Rosa26 locus, and contains, from 5' to 3', the following: (i) 3 ' splicing sequence; and (ii) an NLS-Cas9 coding sequence, and step (b) involves evaluating the modification of a target genomic locus in the liver of a non-human animal. In some such methods, the non-human animal is a mouse, the AAV is AAV8 delivered to the non-human animal by intravenous injection, the Cas expression cassette is operably linked to the endogenous Rosa26 promoter, is inserted into the first intron of the Rosa26 locus, and contains 5' to 3 ' the following: (i) 3' splicing sequence; and (ii) an NLS-Cas9 coding sequence, and step (b) involves evaluating the modification of a target genomic locus in the liver of a non-human animal.

[0024] In one aspect, methods are provided for optimizing the ability of a CRISPR/Cas nuclease to modify a target genomic locus in vivo. Some such methods include: (i) performing any of the above methods of testing the ability of a CRISPR/Cas nuclease to modify a target genomic locus in vivo for the first time in a first non-human animal; (II) changing the variable and performing the method of step (I) a second time with the changed variable in a second non-human animal; and (III) comparing the modification of the target genomic locus in step (I) with the modification of the target genomic locus in step (II), and selecting a method resulting in a modification of the target genomic locus having one or more of the following: increased efficiency, increased accuracy, increased consistency, or increased specificity.

[0025] According to some such methods, the altered variable in step (II) is an AAV serotype. In some such methods, the altered variable in step (II) is the route of administration for administering the guide RNA to a non-human animal. In some such methods, the altered variable in step (II) is the concentration or amount of guide RNA administered to the non-human animal. In some such methods, the altered variable in step (II) is a guide RNA (eg, the shape or sequence of the guide RNA) administered to a non-human animal. In some such methods, the method comprises administering an exogenous donor nucleic acid, wherein the variable in step (II) is a delivery method for administering the exogenous donor nucleic acid to a non-human animal. According to some such methods, the method involves administering an exogenous donor nucleic acid, and the altered variable in step (II) is the route of administration for administering the exogenous donor nucleic acid to a non-human animal. In some such methods, the method involves administering an exogenous donor nucleic acid, and the variable in step (II) is the concentration or amount of the exogenous donor nucleic acid administered to the non-human animal. In some such methods, the method involves administering an exogenous donor nucleic acid, and the variable in step (II) is the concentration or amount of guide RNA administered to the non-human animal relative to the concentration or amount of exogenous donor nucleic acid administered to the non-human animal. . In some such methods, the altered variable in step (II) is an exogenous donor nucleic acid (eg, a form of the exogenous donor nucleic acid) administered to a non-human animal.

Brief description of the figures

[0026] Figure 1 shows the Cas9 allele (MAID2599; not to scale) containing from 5' to 3' the following: 3' splicing sequence; loxP first site, neomycin resistance gene; polyadenylation signal; second loxP site; encoding NLS-Cas9 sequence; a P2A peptide coding sequence; and a GFP coding sequence.

[0027] Figure 2A shows NHEJ activity in F1H4 mouse wild-type embryonic stem cells (mESCs) and Cas9-expressing mESCs with and without lox-stop-lox neomycin cassette (MAID2599 and MAID2600, respectively) following administration of two sgRNAs (plasmid or as RNA) targeting the start and stop codon regions of the first target gene, optionally in combination with the introduction of the Cas9 plasmid. The efficiency of the 5' cut was measured in the left panel, the efficiency of the 3' cut was measured in the middle panel, the frequency with which the intermediate DNA was completely deleted was measured in the right panel.

[0028] Figure 2B shows cut efficiency (left panel) and HDR efficiency (right panel) after administration of sgRNA (as plasmid or as RNA) targeted to a second target gene along with single-stranded oligodeoxynucleotide (ssODN) as a point mutation donor , optionally in combination with a Cas9 plasmid.

[0029] Figures 3A-3F show bright-field images of liver (Figure 3A), kidney (Figure 3B) and brain (Figure 3C) tissues from wild-type and heterozygous Cas9 expressing mice (MAID2600) and GFP-derived fluorescence images. liver (FIG. 3D), kidney (FIG. 3E), and brain (FIG. 3F) tissues from wild type and Cas9 expressing mice (MAID2600).

[0030] Figure 4A shows Cas9 mRNA expression levels in various tissues isolated from heterozygous Cas9 expressing mice (MAID2600) as determined by RT-qPCR. The y-axis shows delta Ct + 1 compared to the mean Ct Cas9 from brain tissue.

[0031] Figure 4B shows Cas9 protein expression in various tissues isolated from wild type and heterozygous Cas9 expressing mice (MAID2600). Actin was used as a control.

[0032] Figure 4C shows mean expression levels of Cas9 mRNA and beta-2-microglobulin (B2m) in various tissues isolated from heterozygous Cas9 expressing mice (MAID2600) as determined by RT-qPCR. The number of samples tested from each type of tissue is indicated above the bars.

[0033] Figures 5A-5B show percent NHEJ activity (indel frequency) in the third target gene (target gene 3) in primary hepatocytes isolated from wild-type mice (Figure 5A) and cassette-deleted Cas9 mice (MAID2600; Figure 5B). ) after lipid nanoparticle (LNP) delivery of any of the following: GFP mRNA and control (dead) sgRNA, GFP mRNA and sgRNA target gene 3, or Cas9 mRNA and sgRNA target gene 3. mRNA concentrations of 15.6, 62, 5, 250 and 1000 ng/ml.

[0034] Figures 6A-6D show the serum levels of a protein that is secreted by the liver and found in serum and encoded by a third target gene (target gene 3) after sgRNA of the target gene was administered to 3 wild-type (msCas9 -) mice or Cas9-expressing mice with deletion cassette (msCas9+; MAID2600) via hydrodynamic DNA delivery (HDD), lipid nanoparticle (LNP) delivery, or adeno-associated virus (AAV) delivery via tail vein injection. In some cases, Cas9 is also administered (in mRNA form for LNP delivery and in DNA form for HDD (Cas9 plasmid) and AAV delivery). Untreated mice, LNP control mice, AAV control mice and HDD control mice were used as negative controls. Three groups of mice were tested for LNP-mediated delivery: (1) Cas9 expressing mice (3 males + 3 females; 2 mg/kg control guide RNA + GFP mRNA); (2) Cas9 expressing mice (3 males + 3 females; 2 mg/kg guide RNA for target gene 3 + GFP mRNA); and (3) WT mice (3 males + 3 females; 2 mg/kg guide RNA for target gene 3 + Cas9 mRNA). Two groups of mice were tested for AAV-mediated delivery: (1) Cas9 expressing mice (3 males + 3 females; AAV8 guide RNA for target gene 3); and (2) WT mice (3 males + 3 females; AAV8 guide RNA for gene target 3 + AAV8-Cas9). Two groups of mice were tested for HDD: (1) Cas9 expressing mice (3 males + 3 females; guide RNA for target gene 3); and (2) WT mice (3 males + 3 females; target gene guide RNA 3 + Cas9). Serum levels of target gene 3 protein were measured in male mice (Figures 6A and 6B) and female mice (Figures 6C and 6D) and were measured on day 7 (Figures 6A and 6C) and day 21 (Figures 6B and 6D).

[0035] Figure 7 shows the percent NHEJ activity (indel frequency) at the target gene 3 locus in the liver of wild-type (msCas9 -) and cassette-deleted Cas9 mice (msCas9 +; MAID2600) one month after delivery with lipid nanoparticles (LNP) of sgRNA alone or together with Cas9 mRNA, hydrodynamic delivery (HDD) of sgRNA plasmid alone or together with Cas9 plasmid or AAV8-sgRNA alone or together with AAV8-Cas9.

[0036] Figure 8A shows percent NHEJ activity (indel frequency) at the target gene 4 locus in the liver of cassette-deleted Cas9 (MAID2600) mice 3-4 weeks after AAU8 delivery of sgRNA by tail vein injection. UNT = untreated control.

[0037] Figure 8B shows the relative expression levels of target gene 4, as determined by TAQMAN analysis, in liver tissue isolated from cassette-deleted Cas9 (MAID2600) mice 3-4 weeks after AAU8 delivery of sgRNA by tail injection. vein. The WT master mix refers to all five sgRNA viruses mixed together and injected into wild-type mice as a negative control.

[0038] Figure 9 shows a Western blot of Cas9 expression in LSL-Cas9 (MAID2599) mice in liver, spleen and kidney samples isolated one week after LNP-Cre was injected into the tail vein. Mice without LNP-Cre injections were used as negative controls. Cas9 mice with cassette removed (MAID2600) were used as positive controls.

[0039] Figure 10 shows serum levels of a protein that is secreted by the liver and found in serum and encoded by the third target gene (target gene 3) 1 week and 3 weeks after injection of sgRNA target gene 3 into LSL-Cas9 (MAID2599) mice by AAV8 , either alone or together with LNP-Cre. Mice without either LNP-Cre or AAV8-rPHK were used as negative controls. All conditions were also tested in cassette-deleted mice (ROSA Cas9; MAID2600).

[0040] Figure 11 shows percent NHEJ activity (indel frequency) at target gene 3 locus in liver samples isolated 3 weeks after injection of target gene 3 sgRNA into LSL-Cas9 (MAID2599) mice with AAV8, either alone or together with LNP Cre. Mice without either LNP-Cre or AAV8-rPHK administration were used as negative controls. All conditions were also tested in cassette-deleted mice (ROSA Cas9; MAID2600).

[0041] Figure 12A shows a Western blot for Cas9 in liver samples isolated from LSL-Cas9 (MAID2599) mice and LSL-Cas9/Alb-Cre mice. Actin was used as a load control.

[0042] Figure 12B shows a Western blot for Cas9 in brain samples isolated from LSL-Cas9 (MAID2599) mice and LSL-Cas9/Alb-Cre mice. Actin was used as a load control.

[0043] Figure 13 shows serum levels of a protein that is secreted by the liver and found in serum and encoded by a third target gene (target gene 3) 1 week after sgRNA injection of target gene 3 into WT mice, cassette-deleted Cas9 mice (ROSA Cas9; MAID2600), LSL-Cas9 mice (MAID2599), Alb-Cre mice, or LSL-Cas9/Alb-Cre mice via AAV8.

[0044] Figure 14 shows four different Cas9 alleles (not to scale), including MAID2599 allele (MAID2600 after lox-stop-lox (LSL) cassette removal), MAID2658 allele (MAID2659 after LSL cassette removal), MAID2660 allele ( MAID2661 after LSL cassette removal) and MAID2672 allele (MAID2673 after LSL cassette removal).

Definitions

[0045] As used interchangeably herein, the terms "protein", "polypeptide", and "peptide" include polymeric forms of amino acids of any length, including encoded and non-encoded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides characterized by modified peptide backbones.

[0046] Proteins are considered to have an "N-terminus" and a "C-terminus". The term "N-terminus" refers to the beginning of a protein or polypeptide ending in an amino acid with a free amino group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (of a protein or polypeptide) ending in a free carboxyl group (-COOH).

[0047] Used interchangeably herein, the terms "nucleic acid" and "polynucleotide" include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. These include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other naturally occurring, chemically modified, biochemically modified, non-naturally occurring, or derivatized nucleotide bases.

[0048] Nucleic acids are thought to have "5' ends" and "3' ends" because mononucleotides react to form oligonucleotides in such a way that the 5'-phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction through a phosphodiester bond. The end of an oligonucleotide is called the "5' end" if its 5' phosphate is not bonded to the 3' oxygen of the mononucleotide pentose ring. The end of an oligonucleotide is called the "3' end" if its 3' oxygen is not bonded to the 5' phosphate of another mononucleotide pentose ring. It can be said that the nucleic acid sequence, even if it is located within a larger oligonucleotide, is characterized by the presence of 5' and 3' ends. In a linear or circular DNA molecule, discrete elements are referred to as "upstream" or 5' from "downstream" or 3' elements.

[0049] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence is integrated into the cell's genome. Any protocol can be used to stably incorporate a nucleic acid into a cell's genome.

[0050] The term "expression vector" or "expression construct" refers to a recombinant nucleic acid containing the desired coding sequence operably linked to the corresponding nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or host organism. Nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operator (optionally) and a ribosome binding site, among other sequences. It is well known that eukaryotic cells use promoters, enhancers, and termination and polyadenylation signals, although some elements may be removed and other elements may be added without compromising the necessary expression.

[0051] The term "targeting vector" refers to a recombinant nucleic acid that can be introduced by homologous recombination, ligation, non-homologous end joining, or any other method of recombination with respect to a target position in the cell's genome.

[0052] The term "viral vector" refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient to or permit packaging into a viral vector particle. The vector and/or particle can be used to transfer DNA, RNA or other nucleic acids into cells ex vivo or in vivo. Numerous forms of viral vectors are known.

[0053] The term "isolated" in relation to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified from other cellular or body components that may normally be present in situ, up to and including a substantially pure preparation of a protein, nucleic acid, or cell. The term "isolated" also includes proteins and nucleic acids that have no naturally occurring counterpart, or proteins or nucleic acids that have been chemically synthesized and thus are not substantially contaminated with other proteins or nucleic acids. The term "isolated" also includes proteins, nucleic acids, or cells that have been separated or purified from most other cellular or body components that naturally accompany them (e.g., other cellular proteins, nucleic acids, or cellular or extracellular components) .

[0054] The term "wild type" includes entities characterized by structure and/or activity found in a normal (as opposed to mutated, diseased, altered, etc.) state or context. Wild-type genes and polypeptides often exist in several different forms (eg, alleles).

[0055] The term "endogenous sequence" refers to a nucleic acid sequence that occurs naturally within a non-human cell or animal. For example, the endogenous Rosa26 sequence of a non-human animal refers to the native Rosa26 sequence that occurs naturally at the Rosa26 locus in a non-human animal.

[0056] "Exogenous" molecules or sequences include molecules or sequences that are not normally present in the cell in that form. Normal presence includes being present at a particular developmental stage and under particular environmental conditions of a cell. For example, an exogenous molecule or sequence may include a mutant version of the corresponding endogenous sequence in the cell, such as a humanized version of the endogenous sequence, or may include a sequence corresponding to the endogenous sequence in the cell, but in a different form (i.e., not within chromosomes). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in such form in a particular cell at a certain developmental stage under certain environmental conditions.

[0057] The term "heterologous" when used in the context of a nucleic acid or protein indicates that the nucleic acid or protein contains at least two segments that do not naturally occur together in the same molecule. For example, the term "heterologous" when used in relation to nucleic acid segments or protein segments indicates that the nucleic acid or protein contains two or more subsequences that are not in the same relationship to each other (eg combined) in nature. As one example, a "heterologous" region of a nucleic acid vector is a nucleic acid segment within or attached to another nucleic acid molecule that is not found in association with another molecule in nature. For example, the heterologous region of a nucleic acid vector may include a coding sequence flanked by sequences that are not naturally found in association with the coding sequence. Similarly, a "heterologous" region of a protein is a segment of amino acids within or attached to another peptide molecule that is not naturally found in association with another peptide molecule (eg, a fusion protein or a tagged protein). Similarly, the nucleic acid or protein may contain a heterologous tag or a heterologous secretion sequence or a heterologous localization sequence.

[0058] "Codon optimization" takes advantage of the degeneracy of codons, which manifests itself in the multiplicity of combinations of three-base-pair codons that define an amino acid, and typically involves the process of modifying a nucleic acid sequence for increased expression in specific host cells by replacing at least one codon of the native sequence with a codon that is most or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a Cas9 protein can be modified to replace codons having a higher frequency of use in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a cell rodent, mouse cell, rat cell, hamster cell or any other host cell compared to a naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in the "Codon Usage Database". These tables can be adapted in several ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available to optimize codons of a particular sequence for expression in a particular host (see, for example, Gene Forge).

[0059] A "promoter" is a DNA regulatory region, typically containing a TATA box, capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. The promoter may additionally contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein modulate the transcription of an operably linked polynucleotide. A promoter may be active in one or more of the cell types disclosed herein (e.g., eukaryotic cell, non-human mammalian cell, human cell, rodent cell, pluripotent cell, single cell embryo, differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-limited promoter (eg, a developmental stage promoter), or a spatially-limited promoter (eg, a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in International Patent Publication WO 2013/176772, incorporated herein by reference in its entirety for all purposes.

[0060] A constitutive promoter is a promoter that is active in all tissues or specific tissues at all stages of development. Examples of constitutive promoters include human cytomegalovirus (hCMV) immediate early promoter, mouse cytomegalovirus (mCMV) immediate early promoter, human elongation factor 1 alpha (hEF1α) promoter, mouse elongation factor 1 alpha (mEF1α) promoter, mouse phosphoglycerate kinase (PGK) promoter , the chicken beta-actin hybrid (CAG or CBh) promoter, the SV40 early promoter, and the tubulin beta 2 promoter.

[0061] Examples of inducible promoters include, for example, chemically controlled promoters and physically controlled promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (eg, the alcohol dehydrogenase (a1cA) gene promoter), tetracycline-regulated promoters (eg, tetracycline-sensitive promoter, tetracycline operator sequence (tetO), tet-On promoter, or tet-Off promoter ), steroid-regulated promoters (eg, rat glucocorticoid receptor, estrogen receptor promoter, or ecdysone receptor promoter), or metal-regulated promoters (eg, metalloprotein promoter). Physically regulated promoters include, for example, temperature regulated promoters (eg heat shock promoter) and light regulated promoters (eg light induced promoter or light repressed promoter).

[0062] Tissue-specific promoters can be, for example, neuron-specific promoters, glia-specific promoters, muscle cell-specific promoters, heart cell-specific promoters, kidney cell-specific promoters, specific for bone cell promoters, endothelial cell-specific promoters, or immune cell-specific promoters (eg B cell promoter or T cell promoter).

[0063] Developmentally regulated promoters include, for example, promoters active only during the embryonic stage of development or only in the adult cell.

[0064] The term "operably linked" or "operably linked" includes the contiguous position of two or more components (e.g., a promoter and another element of the sequence) such that both components function normally and provide the possibility that at least one of the components may mediate a function that is acted upon by at least one of the other components. For example, a promoter may be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. The operative linkage may include sequences that are adjacent to each other or act in trans (eg, a regulatory sequence may act at a distance to direct transcription of a coding sequence).

[0065] The term "complementarity" of nucleic acids means that a nucleotide sequence in one nucleic acid strand, due to the orientation of its nucleic base groups, forms hydrogen bonds with another sequence on the opposite nucleic acid strand. Complementary bases in DNA are typically A with T and C with G. In RNA they are typically C with G and U with A. Complementarity may be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids means that two nucleic acids can form a duplex, in which each base in the duplex is linked to a complementary base by a Watson-Crick pairing. "Substantial" or "sufficient" complementarity means that the sequence in one strand is not fully and/or perfectly complementary to the sequence in the opposite strand, but that sufficient binding occurs between bases on the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted using sequences and standard mathematical calculations to predict the Tm (melting point) of the hybridized strands, or by empirically determining the Tm using routine methods. Tm includes the temperature at which the population of hybridization complexes formed between two nucleic acid strands is 50% denatured (ie, the population of double-stranded nucleic acid molecules becomes half dissociated into single strands). Below Tm, formation of the hybridization complex is preferred, while above Tm, melting or strand separation in the hybridization complex is preferred. The Tm can be estimated for a nucleic acid having a known G+C content in an aqueous 1 M NaCl solution using, for example, Tm=81.5+0.41 (% G+C), although other known Tm calculations take into account the structural characteristics of the nucleic acid. acids.

[0066] "Hybridization condition" includes a total environment in which one nucleic acid strand binds to a second nucleic acid strand through the interaction of complementary strands and hydrogen bonding to form a hybridization complex. Such conditions include chemical components and their concentrations (eg, salts, chelating agents, formamide) of an aqueous or organic solution containing nucleic acids, and the temperature of the mixture. Other factors such as incubation time or reaction chamber size may affect the environment. See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), incorporated herein by reference in its entirety for all purposes.

[0067] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases are possible. Conditions suitable for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables that are well known. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value for nucleic acid hybrids characterized by those sequences. For hybridization between nucleic acids with short complementarity regions (e.g., complementarity of more than 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides), the position of mismatches becomes important (see Sambrook et al. ., previously, 11.7-11.8). Typically, the length of the nucleic acid to be hybridized is at least about 10 nucleotides. Illustrative minimum lengths for a hybridizable nucleic acid include at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides. In addition, the temperature and salt concentration of the wash solution can be adjusted as necessary, according to factors such as the length of the complementarity region and the degree of complementarity.

[0068] A polynucleotide sequence need not be 100% complementary to the target nucleic acid sequence in order to be specifically hybridizable. In addition, the polynucleotide can hybridize to one or more segments such that intermediate or adjacent segments do not participate in the hybridization event (eg, loop structure or hairpin structure). A polynucleotide (e.g., gRNA) may have at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to the target region within the target region. the nucleic acid sequence they target. For example, an gRNA in which 18 out of 20 nucleotides are complementary to the target region and therefore will hybridize specifically will represent 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be adjacent to each other or to complementary nucleotides.

[0069] Percentage complementarity between specific regions of nucleic acid sequences within nucleic acids can be determined in the usual manner using BLAST programs (Basic Local Alignment Search Tools) and PowerBLAST programs (Altschul et al. (1990) J. Mol. Biol. 215: 403-410; Zhang and Madden (1997) Genome Res. 7: 649-656) or using the Gap program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using the default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489).

[0070] Many different components are used in the methods and compositions provided herein. Some components throughout the present description may be characterized by the presence of active variants and fragments. Such components include, for example, Cas proteins, CRISPR RNA, tracrPHK, and guide RNAs. The biological activity for each of these components is described elsewhere herein. The term "functional" refers to the innate ability of a protein or nucleic acid (or fragment or variant thereof) to exhibit biological activity or function. Such biological activities or functions may include, for example, the ability of the Cas protein to bind to the guide RNA and to the target DNA sequence. The biological functions of the functional fragments or variants may be the same or may actually be altered (eg, in terms of their specificity, selectivity, or potency) from their original counterparts, but retaining the underlying biological function.

[0071] The term "variant" refers to a nucleotide sequence different from the sequence most common in the population (eg, by one nucleotide), or a protein sequence different from the sequence most common in the population (eg, by one amino acid).

[0072] The term "fragment" in relation to a protein means a protein that is shorter or contains fewer amino acids than a full-length protein. The term "fragment" in relation to a nucleic acid means a nucleic acid that is shorter or contains fewer nucleotides than a full-length nucleic acid. A fragment may be, for example, an N-terminal fragment (ie removal of part of the C-terminal end of the protein), a C-terminal fragment (ie removal of part of the N-terminal end of the protein), or an internal fragment.

[0073] The term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in two sequences that are the same when aligned for maximum match in a specified comparison window. When percent sequence identity is used with proteins, positions of residues that are not identical are often distinguished by conservative amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. . When sequences differ in conservative substitutions, the percent sequence identity can be increased to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to be characterized by "sequence similarity" or "similarity". Means for such adjustment are well known. Typically, this includes evaluating a conservative substitution as a partial rather than a complete mismatch, which increases the percent sequence identity. Thus, for example, when an identical amino acid scores 1 and a non-conservative substitution scores 0, a conservative substitution scores from 0 to 1. Conservative substitution scores are calculated, for example, as implemented in the PC/GENE program (Intelligenetics, Mountain View, California ).

[0074] "Percent sequence identity" includes a value determined by comparing two optimally aligned sequences (highest number of perfectly matched residues) in the comparison window, wherein a portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps ) compared to a reference sequence (which contains no addition or deletion) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. . Unless otherwise noted (eg, the shorter sequence includes an associated heterologous sequence), the comparison window is the full length of the shorter of the two compared sequences.

[0075] Unless otherwise indicated, sequence identity/similarity values include the value obtained using GAP version 10 using the following parameters: % identity and % similarity for the nucleotide sequence using a GAP gap penalty of 50, and the gap lengthening penalty of 3 and the replacement matrix nwsgapdna. cmp; % identity and % similarity for amino acid sequence using GAP gap penalty of 8 and gap extension penalty of 2 and substitution matrix BLOSUM62; or any equivalent program. An "equivalent program" includes any sequence comparison program that, for any two sequences under consideration, generates an alignment characterized by identical nucleotide or amino acid residue matches and an identical percentage of sequence identity compared to the corresponding alignment generated by GAP version 10.

[0076] The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in a sequence with another amino acid of similar size, charge, or polarity. Examples of conservative substitutions include replacing a non-polar (hydrophobic) moiety such as isoleucine, valine, or leucine with another non-polar moiety. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. In addition, substitution of a basic residue such as lysine, arginine or histidine with another, or substitution of one acidic residue such as aspartic acid or glutamic acid with another acidic residue are further examples of conservative substitutions. Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and/or a polar residue with non-polar residue. Typical classifications of amino acids are shown in Table 1 below.

[0078] The term "in vitro" includes artificial environments and processes or reactions that occur in an artificial environment (eg, in a test tube). The term "in vivo" includes natural environments (eg, cell, organism or organism) and processes or reactions that occur in the natural environment. The term "ex vivo" includes cells that have been removed from the human body, as well as processes or reactions that occur in such cells.

[0079] The term "reporter gene" refers to a nucleic acid with a sequence encoding a gene product (typically an enzyme) that is readily and quantitatively analyzed when the construct containing the reporter gene sequence is operably linked to an endogenous or heterologous promoter and/or enhancer element introduced into cells containing (or which may contain) the factors necessary for the activation of promoter and/or enhancer elements. Examples of reporter genes include, but are not limited to, genes encoding beta-galactosidase (lacZ), genes for bacterial chloramphenicol acetyltransferase (feline), firefly luciferase genes, genes encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. "Reporter protein" refers to a protein encoded by a reporter gene.

[0080] As used herein, the term "fluorescent reporter protein" means a reporter protein that can be detected based on fluorescence, where the fluorescence can be either directly from the reporter protein, the activity of the reporter protein on a fluorogenic substrate, or a protein with an affinity for binding to a fluorescent labeled compound. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, emerald green, Azami Green, monomeric Azami Green, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, citrine, Venus, YPet, PhiYFP, and ZsYellowl), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, azurite, mKalamal, GFPuv, sapphire, and T-sapphire), blue fluorescent proteins (e.g., CFP, eCFP, sky blue, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins (e.g., RFP, mKate, mKate2, mPlum, DsRed-monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611 , mRaspberry, mStrawberry and Jred), orange fluorescent proteins (e.g. mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine and tdTomato) and any other suitable fluorescent protein whose presence in cells can be detected using flow cytometry methods.

[0081] Recovery in response to double-strand breaks (DSB) occurs primarily through two conservative DNA repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22: 886-897, incorporated herein by reference in its entirety for all purposes. Similarly, restoration of a target nucleic acid mediated by an exogenous donor nucleic acid may include any process of exchanging genetic information between two polynucleotides.

[0082] The term "recombination" includes any process of exchange of genetic information between two polynucleotides and can occur by any mechanism. Recombination can occur via homologous repair (HDR) or homologous recombination (HR). HDR or HR involve a form of nucleic acid repair that may require nucleotide sequence homology, uses a "donor" molecule as a template to repair a "target" molecule (i.e. one that has experienced a double strand break), and results in the transfer of a genetic information from donor to target. While not wishing to be bound by any particular theory, such transfer may include correcting the heteroduplex DNA mismatch that forms between the damaged target and the donor, and/or strand synthesis dependent annealing, in which the donor is used to resynthesize the genetic information that will become part of the target and/or associated processes. In some cases, a donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide is integrated into the target DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al. (2012) FLOS ONE 7:e45768:1-9; and Wang et al. (2013) Nat Biotechnol. 31:530-532, each of which is hereby incorporated by reference in its entirety for all purposes.

[0083] NHEJ involves repairing double-strand breaks in a nucleic acid by directly ligating the ends of the breaks to each other or to an exogenous sequence without the need for a homologous template. Ligation of non-contiguous sequences with NHEJ can often result in deletions, insertions, or translocations near the double-strand break site. For example, NHEJ can also result in targeted integration of the exogenous donor nucleic acid by direct ligation of the break ends to the ends of the exogenous donor nucleic acid (ie, NHEJ-based capture). Such NHEJ-mediated targeted integration may be preferred for exogenous donor nucleic acid insertion when homologous repair (HDR) pathways are not easily exploited (eg, in non-dividing cells, primary cells, and cells that perform poorly on homology-based DNA repair). Furthermore, in contrast to homologous repair, knowledge of the relatively large regions of sequence identity flanking the cleavage site (outside of the drooping ends created by Cas-mediated cleavage) is not required, which may be advantageous when attempting targeted insertion into organisms having genomes for which knowledge of the genomic sequence is limited. Integration can occur by blunt-end ligation between the exogenous donor nucleic acid and the cleaved genomic sequence, or by ligation of sticky ends (i.e., characterized by 5' or 3' hanging ends) using an exogenous donor nucleic acid that is flanked by hanging ends that are compatible with those created by the Cas protein in the cleaved genomic sequence. See, for example, US 2011/020722, WO 2014/033644, WO 2014/089290 and Maresca et al. (2013) Genome Res. 23(3):539-546, each of which is incorporated herein by reference in its entirety for all purposes. If the blunt ends are ligated, it may be necessary to excise the target and/or donor to create regions of microhomology necessary for joining the fragments, which may lead to undesirable changes in the target sequence.

[0084] Compositions or methods "providing" or "comprising" one or more of the listed elements may include other elements that are not specifically indicated. For example, a composition that "comprises" or "comprises" a protein may contain the protein alone or in combination with other ingredients. The transitional phrase "consisting essentially of" means that the scope of the claims is to be interpreted as covering the specified elements listed in the claims and elements that do not materially affect the essential(s) and novel feature(s) of the claimed invention. . Thus, it is not intended that the term "consisting essentially of" when used in the claims should be interpreted as equivalent to "comprising".

[0085] "Optional" or "optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases in which the event or circumstance occurs and cases in which it does not occur.

[0086] The designation of a range of values includes all integers within the range or defining a range and all subranges defined by integers within the range

[0087] Unless otherwise clear from the context, the term "approximately" encompasses values within a standard error margin of measurement (eg, SEM) of a specified value.

[0088] The term "and/or" refers to and encompasses any possible combinations of one or more of the related listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0089] The term "or" refers to any one member of a particular list, and also includes any combination of members of this list.

[0090] Singular forms include plural forms unless the context clearly dictates otherwise. For example, the term "Cas protein" or "at least one Cas protein" may include a plurality of Cas proteins, including mixtures thereof.

[0091] Statistically significant means p ≤ 0.05.

Detailed disclosure of the present invention

I. Overview

[0092] The CRISPR/Cas9 system is a powerful tool for genome engineering. One of the limitations of the in vivo system is the need for simultaneous introduction of all components into a living organism. A typical way to introduce these components is to transiently transfect the DNA constructs into cells that will generate the appropriate RNA and protein. While this approach is effective, it has an inherent disadvantage as cells must rely on plasmid DNA constructs to first undergo transcription and then translation before the Cas9 protein becomes available to interact with the sgRNA component. Better methods and tools are needed to better assess the activity of CRISPR/Cas agents and to evaluate different delivery methods and parameters to target specific tissues or cell types in vivo.

[0093] Provided herein are methods and compositions for assessing the activity of CRISPR/Cas-mediated non-homologous end joining (NHEJ) and/or CRISPR/Cas-induced recombination of a target genomic locus with an exogenous donor nucleic acid in vivo and ex vivo. The methods and compositions use cells and human animals containing a Cas expression cassette (e.g., a genomically integrated Cas expression cassette) such that the Cas protein can be constitutively available or, for example, available in a tissue-specific or time-specific manner.

[0094] Non-human animals containing Cas expression cassettes simplify the process of testing the delivery and activity of CRISPR/Cas components in vivo, since only guide RNAs need be administered to a non-human animal. In addition, Cas expression cassettes may optionally be conditional Cas expression cassettes that may be selectively expressed in certain tissues or at certain developmental stages, thereby reducing the risk of Cas-mediated toxicity in vivo, or may be constitutively expressed to allow activity to be tested in any and all all types of cells, tissues and organs.

[0095] In addition, methods and compositions are provided for obtaining and using these non-human animals to test and measure the ability of CRISPR/Cas nuclease to modify a target genomic locus in vivo. In some such methods for testing and measuring the ability of a CRISPR/Cas nuclease to modify a target genomic locus in vivo, the guide RNA can be delivered to a Cas expressing non-human animal via AAV-mediated delivery. As shown in Example 1, AAV-mediated delivery of guide RNAs to Cas9 expressing mice, and in particular AAV8-mediated delivery to the liver, results in surprisingly higher levels of CRISPR/Cas targeting than LNP or HDD delivery of guide RNAs to Cas9 expressing mice or delivery of both Cas9 and guide RNAs to wild-type mice.

II. Non-human animals containing Cas expression cassettes

[0096] The methods and compositions disclosed herein use non-human animals or cells containing Cas expression cassettes to evaluate the capacity of clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems or components of such systems ( for example, guide RNAs introduced into the body of a non-human animal or cell) to modify the target genomic locus in vivo or ex vivo.

[0097] CRISPR/Cas systems include transcripts and other elements involved in the expression or control of Cas gene activity. The CRISPR/Cas system may be, for example, a type I, type II, or type III system. Alternatively, the CRISPR/Cas system may be a type V system (eg, subtype V-A or subtype V-B). The CRISPR/Cas systems used in the compositions and methods disclosed herein may not be naturally occurring. A "non-naturally occurring" system includes anything that indicates human involvement, for example, one or more components of the system are altered or mutated from their natural state, while at least substantially free of at least one other component, with which they are naturally associated in nature, or associated with at least one other component with which they are not naturally associated. For example, non-naturally occurring CRISPR/Cas systems may use CRISPR complexes containing gRNA and Cas protein that do not naturally occur together, Cas protein that does not occur naturally, or gRNA that does not occur naturally.

[0098] The methods and compositions disclosed herein use CRISPR/Cas systems by testing the ability of CRISPR complexes (comprising a guide RNA (gRNA) in complex with a Cas protein) to induce site-directed cleavage events at a target genomic locus in vivo to modify the target genomic locus by non-homologous end joining (NHEJ), by homologous repair in the presence of an exogenous donor nucleic acid, or by any other repair or recombination techniques.

A. Cas9 Expressing Non-Human Animals

[0099] Cells and non-human animals disclosed herein contain the Cas expression cassette. Cas proteins typically contain at least one RNA recognition or binding domain that can interact with guide RNAs (gRNAs, described in more detail below) and nuclease domains. The nuclease domain has catalytic activity for nucleic acid cleavage, which involves breaking the covalent bonds of the nucleic acid molecule. Cleavage may result in blunt or staggered ends and may be single or double stranded. The Cas protein may have full cleavage activity to create a double-strand break in the target nucleic acid (eg, a blunt-ended double-strand break), or may be a nickase that creates a single-strand break in the target nucleic acid.

[00100] Cells or non-human animals containing the Cas expression cassette have the advantage that they require only the delivery of guide RNAs to detect CRISPR/Cas-mediated modification of the target genomic locus.

(1) Cas expression cassettes

[00101] The cells and non-human animals described herein contain the Cas expression cassette. The Cas expression cassette may be stably integrated into the genome (ie, chromosome) of a cell or non-human animal, or it may be located outside the chromosome (eg, extrachromosomally replicating DNA). Optionally, the Cas expression cassette is stably integrated into the genome. A stably integrated Cas expression cassette may be randomly integrated into the genome of a non-human animal (ie, transgenic), or it may be integrated into a predetermined region of the genome of a non-human animal (ie, knockin). Optionally, the Cas expression cassette is stably integrated into the safe harbor locus as described elsewhere herein. The target genomic locus at which the Cas expression cassette is stably integrated may be heterozygous for the Cas expression cassette or homozygous for the Cas expression cassette.

[00102] The Cas protein encoded by the Cas expression cassette can be any Cas protein (eg, Cas9 protein), examples of which are described below. The encoded Cas protein may further comprise one or more nuclear localization signals (NLS) (e.g., N-terminal NLS and C-terminal NLS), and the sequence encoding the Cas protein may be codon-optimized for a cell or non-human animal, such as described below. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical to the sequence of the Cas9 protein shown in SEQ ID NO: 19. The coding sequence may contain, consist essentially of, or consist of the sequence of at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100% identical to the Cas9 coding sequence shown in SEQ ID NO: 30.

[00103] An exemplary Cas expression cassette contains a Cas coding sequence downstream of a polyadenylation signal or transcription terminator flanked by recombinase recognition sites recognized by a site-specific recombinase. The polyadenylation signal or transcription terminator prevents transcription and expression of the Cas protein. However, after exposure to a site-specific recombinase, the polyadenylation signal or transcription terminator will be excised and the Cas protein can be expressed.

[00104] Such a configuration for a Cas expression cassette can provide tissue-specific or developmental-specific expression in non-human animals containing the Cas expression cassette if the polyadenylation signal or transcription terminator is excised in a tissue-specific or developmental-specific manner. This can reduce toxicity due to prolonged expression of the Cas protein in a cell or body of a non-human animal, or expression of the Cas protein at undesirable developmental stages or in undesired cell or tissue types of a non-human animal. For example, toxicity can result from the breakdown and destruction of non-target sites. See, for example, Parikh et at. (2015) PLoS One 10(1):e0116484. Inducible expression may also be beneficial, as the ability to edit certain genes in certain tissues (such as immune cells, for example) can be detrimental as well as potentially trigger an immune response. For example, in some cases, if a gene is mutated throughout an individual's body, it may be lethal, but if it mutates in a specific type of tissue or cell, it will be beneficial. Excision of the polyadenylation signal or transcription terminator can be achieved in a tissue-specific or developmental-stage-specific manner if the non-human animal containing the Cas expression cassette further comprises a site-specific recombinase operably linked to a tissue-specific or developmental-stage-specific promoter (e.g., promoter albumin for liver-specific expression or insulin-2 promoter for pancreas-specific expression). Similarly, LNP formulations specific to the liver or other tissues can be used to deliver the recombinase, or AAV delivery methods or tissue-specific AAV serotypes (e.g., AAV8 for the liver or AAV direct injection for the pancreas) can be used to deliver the recombinase. glands). The polyadenylation signal or transcription terminator is then cut out only in these tissues or at these developmental stages, allowing tissue- or developmental-specific expression of the Cas protein to occur. In one example, the Cas protein may be expressed in a liver-specific manner. Examples of such promoters that have been used to develop such lines of non-human animals in which DNA is excised by a recombinase ("recombinase deleter") are disclosed elsewhere herein.

[00105] Any transcription terminator or polyadenylation signal can be used. The term "transcriptional terminator" as used herein refers to a DNA sequence that causes the termination of transcription. In eukaryotes, transcription terminators are recognized by protein factors, and termination is followed by polyadenylation, the process of adding a poly(A) tail to mRNA transcripts in the presence of poly(A) polymerase. The mammalian poly(A) signal typically consists of a core sequence of approximately 45 nucleotides in length, which may be flanked by various accessory sequences that serve to enhance the efficiency of cleavage and polyadenylation. The core sequence consists of a highly conserved upstream element (AATAAA or AAUAAA) in mRNA called the poly-A recognition motif or poly-A recognition sequence) recognized by cleavage and polyadenylation-specific factor (CPSF) and a poorly defined downstream region (rich in Us or Gs and Us) associated with the cleavage stimulating factor (CstF). Examples of transcription terminators that can be used include, for example, human growth hormone (HGH) polyadenylation signal, simian virus 40 (SV40) late polyadenylation signal, rabbit beta-globin polyadenylation signal, bovine growth hormone (BGH) polyadenylation signal , a phosphoglycerate kinase (PGK) polyadenylation signal, an AOX1 transcription termination sequence, a CYC1 transcription termination sequence, or any transcription termination sequence known to be suitable for regulating gene expression in eukaryotic cells.

[00106] Site-specific recombinases include enzymes that can facilitate recombination between recombinase recognition sites where two recombination sites are physically separated in the same nucleic acid or in separate nucleic acids. Examples of recombinases include Cre, Flp and Dre recombinases. One example of a Cre recombinase gene is Crei, in which two exons encoding Cre recombinase are separated by an intron to prevent its expression in a prokaryotic cell. Such recombinases may additionally contain a nuclear localization signal to facilitate nuclear localization (eg, NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by a site-specific recombinase and can serve as a substrate for a recombination event. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2 and lox5171.

[00107] The Cas expression cassette can be operably linked to any suitable promoter for in vivo expression in a non-human animal. The non-human animal can be any suitable non-human animal as described elsewhere herein. As one example, a Cas expression cassette can be operably linked to an endogenous promoter at a target genomic locus, such as the Rosa26 promoter. Alternatively, the Cas expression cassette can be operably linked to an exogenous promoter, such as a constitutively active promoter (e.g., the CAG promoter or chicken beta-actin promoter/enhancer in combination with the cytomegalovirus (CMV) immediate early enhancer (CAGG)), a conditional promoter that is inducible a promoter, a temporally restricted promoter (eg, a developmentally regulated promoter), or a spatially restricted promoter (eg, a cell-specific or tissue-specific promoter). Such promoters are well known and are discussed elsewhere herein. An exemplary CAGG promoter is shown in SEQ ID NO: 38 or contains, consists essentially of, or consists of the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% identical to the sequence shown in SEQ ID NO: 38.

[00108] The Cas expression cassettes disclosed herein may also contain other components. The Cas expression cassette may further comprise a 3' splicing sequence at the 5' end of the Cas expression cassette and/or a second polyadenylation signal following the coding sequence for the Cas protein at the 3' end of the Cas expression cassette. The Cas expression cassette may further comprise a selection cassette containing, for example, a coding sequence for a drug resistance protein. Examples of suitable selection markers include neomycin phosphotransferase (neo _r ), hygromycin B phosphotransferase (hyg _r ), puromycin N-acetyltransferase (puro _r ), blasticidin S-deaminase (bsr _r ), xanthine/guanine phosphoribosyltransferase (gpt), and viral thymidine kinase. herpes simplex (HSV-k). Optionally, the selection cassette can be flanked by recombinase recognition sites for a site-specific recombinase. If the Cas expression cassette also contains recombinase recognition sites flanking the polyadenylation signal upstream of the Cas coding sequence as described above, optionally a different set of recombinase recognition sites recognized by another recombinase is used to flank the selection cassette. Alternatively, the same set of recombinase recognition sites may flank both the polyadenylation signal to the Cas coding sequence and the selection cassette. An exemplary neo _r -polyadenylation sequence is shown in SEQ ID NO: 37 or contains, consists essentially of, or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO: 37.

[00109] The Cas expression cassette may also contain a nucleic acid encoding a protein tag, such as a 3xFLAG tag. An example of such a tag is provided in SEQ ID NO: 23, which is optionally encoded by SEQ ID NO: 34. For example, the tag may be at the N-terminus of the Cas protein, at the C-terminus of the Cas protein, or within the Cas protein. For example, such an expression cassette can encode a protein containing, consisting essentially of, or consisting of the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical to the 3xFLAG-Cas9 protein sequence shown in SEQ ID NO: 22, or the 3xFLAG-Cas9-P2A-eGFP protein sequence shown in SEQ ID NO: 16. A coding sequence may contain, consist essentially of, or consist of from a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 3xFLAG-Cas9 Cas9 coding sequence shown in SEQ ID NO: 31 or the 3xFLAG-Cas9-P2A-eGFP coding sequence shown in SEQ ID NO: 29, respectively.

[00110] The Cas expression cassette may also contain a nucleic acid encoding one or more reporter proteins, such as a fluorescent protein (eg, green fluorescent protein). Any suitable reporter protein may be used. For example, a fluorescent reporter protein as defined elsewhere herein or a non-fluorescent reporter protein can be used. Examples of fluorescent reporter proteins are presented elsewhere in this document. Non-fluorescent reporter proteins include, for example, reporter proteins that can be used in histochemical or bioluminescent assays, such as beta-galactosidase, luciferase (eg, Renilla luciferase, firefly luciferase, and NanoLuc luciferase), and beta-glucuronidase. The Cas expression cassette may include a reporter protein that can be detected in a flow cytometry assay (e.g., a fluorescent reporter protein such as green fluorescent protein) and/or a reporter protein that can be detected in a histochemical assay (e.g., protein beta-galactosidase). One example of such a histochemical assay is the imaging of histochemical expression of beta-galactosidase in situ by hydrolysis of X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside), which produces a blue precipitate, or using fluorogenic substrates such as as beta-methyl-umbelliferylgalactoside (MUG) and fluorescein digalactoside (FDG).

[00111] The Cas expression cassette in such cases may contain a multicistronic nucleic acid. For example, such nucleic acids may be a Cas protein coding sequence and a reporter protein coding sequence (in any order) separated by an internal ribosome entry intermediate site (IRES) or a 2A peptide intermediate coding sequence. Multicistronic expression constructs simultaneously express two or more separate proteins from the same mRNA (ie, a transcript derived from the same promoter). Suitable strategies for multicistronic protein expression include, for example, the use of a 2A peptide and the use of an internal ribosome entry site (IRES). For example, such nucleic acids may contain coding sequences for two or more reporter proteins separated by an internal ribosome entry intermediate site (IRES) or an intermediate 2A peptide coding sequence. As one example, such multicistronic vectors may use one or more internal ribosome entry sites (IRES) to allow initiation of translation from the interior of the mRNA. As another example, such multicistronic vectors may use one or more 2A peptides. These peptides are small "self-cleaving" peptides, typically 18-22 amino acids long and producing equimolar levels of several genes from the same mRNA. The ribosomes skip the synthesis of the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, resulting in a "cleavage" between the 2A peptide and the peptide immediately below it. See, for example, Kim et al. (2011) PLoS One 6(4): e18556, incorporated herein by reference in its entirety for all purposes. The "cleavage" occurs between the glycine and proline residues found at the C-terminus, meaning that the upstream cistron will have a few extra residues added, while the downstream cistron will start with a proline. As a result, the "cleaved" downstream peptide contains a proline at its N-terminus. 2A-mediated cleavage is a universal phenomenon in all eukaryotic cells. 2A peptides have been identified from picornaviruses, insect viruses, and rotaviruses type C. See, for example, Szymczak et al. (2005) Expert Opin Biol Ther 5:627-638, incorporated herein by reference in its entirety for all purposes. Examples of 2A peptides that can be used include Thosea asigna virus 2A (T2A); 2A porcine teshovirus-1 (P2A); 2A equine rhinitis virus A (ERAV) (E2A) and 2A FMDV (F2A). Exemplary T2A, P2A, E2A, and F2A sequences include: T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 2); P2A (ATNFSLLKQAGDVEENPGP; SEQ ID NO: 3); E2A (QCTNYALLKLAGDVESNPGP; SEQ ID NO: 4); and F2A (VKQTLNFDLLKLAGDVESNPGP; SEQ ID NO: 5). GSG residues can be added to the 5' end of any of these peptides to improve cleavage efficiency. An exemplary coding sequence for P2A with GSG residues added at the 5' end is shown in SEQ ID NO: 32 or contains, consists essentially of, or consists of at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 32. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%o, 99%, or 100% identical to the Cas9-P2A-eGFP eGFP protein sequence shown in SEQ ID NO: 13, or the 3xFLAG-Cas9-P2A-eGFP protein sequence shown in SEQ ID NO: 16. A coding sequence may contain, consist essentially of, or consist of at least 90%, 91%, 92%, 93%, 94%) 95%, 96%, 97%, 98%, 99% or 100% identical Cas9-P2A-eGFP coding sequence shown in SEQ ID N O: 28, or the 3xFLAG-Cas9-P2A-eGFP coding sequence shown in SEQ ID NO: 29, respectively.

[00112] Cas expression cassettes may also contain other elements such as post-transcriptional regulatory elements or polyadenylation signals downstream of the Cas coding sequence. An exemplary post-transcriptional regulatory element is a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) that is represented in SEQ ID NO: 35 or contains, consists essentially of, or consists of the sequence of at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 91%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 35. An exemplary polyadenylation signal is a bovine growth hormone polyadenylation signal as shown in SEQ ID NO: 36 or contains, consists essentially of, or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%, 99%, or 100% of the sequence identical to the sequence shown in SEQ ID NO: 36.

[00113] One exemplary Cas expression cassette contains, from 5' to 3', the following: (a) a 3' splicing sequence; (b) a polyadenylation signal flanked by first and second recombinase recognition sites for recombinase (eg loxP sites for Cre recombinase); (c) a Cas protein coding sequence (eg, an NLS-Cas9 coding sequence, eg with NLS at the N-terminal end and NLS at the C-terminal end); (d) a 2A protein coding sequence (eg, a P2A coding sequence); and (e) a coding sequence for a reporter protein (eg, a fluorescent reporter protein such as green fluorescent protein). See, for example, Figure 1 and MAID2599 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Cas9-P2A-eGFP protein sequence shown in SEQ ID NO: 13.

[00114] Another exemplary expression cassette Cas contains from 5' to 3' the following: (a) 3' splicing sequence; (b) a Cas protein coding sequence (eg, an NLS-Cas9 coding sequence, eg with NLS at the N-terminal end and NLS at the C-terminal end); (c) a 2A protein coding sequence (eg, a P2A coding sequence); and (d) a coding sequence for a reporter protein (eg, a fluorescent reporter protein such as green fluorescent protein). See, for example, MAID2600 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 12. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Cas9-P2A-eGFP protein sequence shown in SEQ ID NO: 13.

[00115] Another exemplary expression cassette Cas contains from 5' to 3' the following: (a) 3' splicing sequence; (b) a polyadenylation signal flanked by first and second recombinase recognition sites for recombinase (eg loxP sites for Cre recombinase); and (c) a Cas protein coding sequence (eg, an NLS-Cas9 coding sequence, eg with NLS at the N-terminal end and NLS at the C-terminal end). See, for example, MAID2660 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 17. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Cas9 protein sequence shown in SEQ ID NO: 19.

[00116] Another exemplary expression cassette Cas contains from 5' to 3' the following: (a) 3' splicing sequence; and (b) a Cas protein coding sequence (eg, an NLS-Cas9 coding sequence, eg with NLS at the N-terminal end and NLS at the C-terminal end). See, for example, MAID2661 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 18. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 91%, 98%, 99%, or 100% identical to the Cas9 protein sequence shown in SEQ ID NO: 19.

[00117] Another exemplary Cas expression cassette contains, 5' to 3', the following: (a) an exogenous promoter (eg, a constitutive promoter such as the CAGG promoter); (b) a polyadenylation signal flanked by first and second recombinase recognition sites for recombinase (eg loxP sites for Cre recombinase); (c) a Cas protein coding sequence (e.g., an NLS-Cas9 coding sequence, e.g., with NLS at the N-terminal end and NLS at the C-terminal end, optionally with a tag at the N-terminal or C-terminal end, such as a 3xFLAG tag at the N-terminal end); (d) a 2A protein coding sequence (eg, a P2A coding sequence); and (e) a coding sequence for a reporter protein (eg, a fluorescent reporter protein such as green fluorescent protein). See, for example, MAID2658 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 14. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 3xFLAG-Cas9-P2A-eGFP protein sequence shown in SEQ ID NO: 16.

[00118] Another exemplary Cas expression cassette contains, 5' to 3', the following: (a) an exogenous promoter (eg, a constitutive promoter such as the CAGG promoter); (b) a Cas protein coding sequence (e.g., an NLS-Cas9 coding sequence, e.g., with NLS at the N-terminal end and NLS at the C-terminal end, optionally with a label at the N-terminal or C-terminal end, such as the tag 3xFLAG at the N-terminal end); (c) a 2A protein coding sequence (eg, a P2A coding sequence); and (d) a coding sequence for a reporter protein (eg, a fluorescent reporter protein such as green fluorescent protein). See, for example, MAID2659 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 15. For example, such an expression cassette can encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 3xFLAG-Cas9-P2A-eGFP protein sequence shown in SEQ ID NO: 16.

[00119] Another exemplary Cas expression cassette contains, 5' to 3', the following: (a) an exogenous promoter (eg, a constitutive promoter such as the CAGG promoter); (b) a polyadenylation signal flanked by first and second recombinase recognition sites for recombinase (eg loxP sites for Cre recombinase); and (c) a Cas protein coding sequence (e.g., an NLS-Cas9 coding sequence, e.g., with NLS at the N-terminal end and NLS at the C-terminal end, optionally with a tag at the N-terminal or C-terminal end, such as a tag 3xFLAG at the N-terminal end). See, for example, MAID2672 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 20. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the 3xFLAG-Cas9 protein sequence shown in SEQ ID NO: 22.

[00120] Another exemplary Cas expression cassette contains, from 5' to 3', the following: (a) an exogenous promoter (eg, a constitutive promoter such as the CAGG promoter); and (b) a Cas protein coding sequence (e.g., an NLS-Cas9 coding sequence, e.g., with NLS at the N-terminal end and NLS at the C-terminal end, optionally with a tag at the N-terminal or C-terminal end, such as a tag 3xFLAG at the N-terminal end). See, for example, MAID2673 in Figure 14. Such an expression cassette may contain, consist essentially of, or consist of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 21. For example, such an expression cassette may encode a protein containing, consisting essentially of, or consisting of an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 3xFLAG-Cas9 protein sequence shown in SEQ ID NO: 22.

[00121] The Cas expression cassettes described herein may be in any form. For example, the Cas expression cassette may be in a vector or plasmid, such as a viral vector. The Cas expression cassette can be operably linked to a promoter in an expression construct capable of directing expression of the Cas protein after removal of the upstream polyadenylation signal. Alternatively, the Cas expression cassette may reside in a targeting vector as defined elsewhere herein. For example, the targeting vector may contain homologous arms flanking the Cas expression cassette, the homologous arms being suitable for directing recombination to the desired target genomic locus to facilitate genomic integration.

[00122] The Cas expression cassettes described herein may be in vitro, they may be within a cell (e.g., an embryonic stem cell) ex vivo (e.g., genomically integrated or extrachromosomal), or they may be in an organism (e.g., a non-human animal ) in vivo (eg, genomically integrated or extrachromosomally). For ex vivo, the Cas expression cassette can be in any type of cell from any organism, such as a totipotent cell such as an embryonic stem cell (e.g. mouse or rat embryonic stem cell) or an induced pluripotent stem cell (e.g. human induced pluripotent stem cell). ). In the in vivo case, the Cas expression cassette can be in any type of organism (eg, non-human animal, as described elsewhere herein below).

(2) Cos proteins and polynucleotides encoding Cas proteins

[00123] Cas proteins typically contain at least one RNA recognition or binding domain that can interact with guide RNAs (gRNAs, described in more detail below). Cas proteins may also contain nuclease domains (eg, DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Some of these domains (eg, DNase domains) may be from the native Cas protein. Other such domains may be added to produce a modified Cas protein. The nuclease domain has catalytic activity for nucleic acid cleavage, which involves breaking the covalent bonds of the nucleic acid molecule. Cleavage may result in blunt or staggered ends and may be single or double stranded. For example, the wild-type Cas9 protein typically creates a blunt-ended cleavage product. Alternatively, a wild-type Cpf1 protein (e.g., FnCpf1) can result in a cleavage product with a 5-nucleotide 5'-pendulous end, with the cleavage occurring after the 18th base pair of the PAM sequence on the off-target strand and after the 23rd base on target chain. The Cas protein may have full cleavage activity to create a double-strand break at the target genomic locus (eg, a blunt-ended double-strand break), or it may be a nickase that creates a single-strand break at the target genomic locus.

[00124] Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10 , Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5 , Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966 and their homologues or modified versions .

[00125] An exemplary Cas protein is a Cas9 protein or a protein derived from Cas9. Cas9 proteins originate from the type II CRISPR/Cas system and generally share four key motifs with conserved architecture. Motifs 1, 2 and 4 are RuvC-like motifs and motif 3 is an HNH motif. Иллюстративные белки Cas9 происходят из Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii , Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilu s, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp. , Acaryochloris marina, Neisseria meningitidis or Campylobacter jejuni. Additional examples of members of the Cas9 family are described in International Patent Publication WO 2014/131833, incorporated herein by reference in its entirety for all purposes. Cas9 from S. pyogenes (SpCas9) (assigned SwissProt accession number Q99ZW2) is an exemplary Cas9 protein. Cas9 from S. aureus (SaCas9) (assigned UniProt accession number J7RUA5) is another exemplary Cas9 protein. Cas9 from Campylobacter jejuni (CjCas9) (assigned UniProt Accession Q0P897) is another exemplary Cas9 protein. See, for example, Kim et al. (2017) Nat. Comm. 8:14500, incorporated herein by reference in its entirety for all purposes. SaCas9 is smaller than SpCas9 and CjCas9 is smaller than SaCas9 and SpCas9.

[00126] Another example of a Cas protein is the Cpf1 protein (CRISPR from Prevotella and Francisella 1). Cpf1 is a large protein (approximately 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding Cas9 domain, along with an analogue of the characteristic arginine-rich Cas9 cluster. However, Cpf1 lacks the HNH nuclease domain that is present in Cas9 proteins, and the RuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9, where it contains long insertions including the HNH domain. See, for example, Zetsche et al. (2015) Cell 163(3):759-771, incorporated herein by reference in its entirety for all purposes. Exemplary Cpf1 proteins are derived from Francisella tularensis 1, Francisella tularensis subsp.novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae. Cpf1 from Francisella novicida U112 (FnCpf1; UniProt accession assigned A0Q7Q2) is an exemplary Cpf1 protein.

[00127] Cas proteins can be wild-type proteins (i.e., proteins that occur naturally), modified Cas proteins (i.e., variants of Cas proteins), or fragments of wild-type proteins or modified Cas proteins. Cas proteins may also be active variants or fragments with respect to the catalytic activity of wild-type Cas proteins or modified Cas proteins. Active variants or fragments in terms of catalytic activity may be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type Cas protein or a modified Cas protein, or a portion thereof, wherein the active variants retain the ability to cut at the desired cleavage site and therefore retain bottom-break or double-strand break-inducing activity. Activity assays for induction of single-strand breaks or induction of double-strand breaks are known and typically measure the overall activity and specificity of the Cas protein on DNA substrates containing a cleavage site.

[00128] Cas proteins can be modified to increase or decrease one or more of the following: nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more of the nuclease domains of the Cas protein may be modified, removed, or inactivated, or the Cas protein may be truncated to remove domains that are not necessary for the function of the protein or for optimization (e.g., to increase or decrease the activity or property of the Cas protein.

[00129] One example of a modified Cas protein is the modified SpCas9-HF1 protein, which is a highly accurate Streptococcus pyogenes variation variant of Cas9 (N497A/R661A/Q695A/Q926A) designed to reduce non-specific DNA contacts. See, for example, Kleinstiver et al. (2016) Nature 529(7587):490-495, incorporated herein by reference in its entirety for all purposes. Another example of a modified Cas protein is a modified version of eSpCas9 (K848A/K1003A/R1060A) designed to reduce unwanted effects. See, for example, Slaymaker et al. (2016) Science 351(6268):84-88, incorporated herein by reference in its entirety for all purposes. Other SpCas9 variants include the K855A and K810A/K1003A/R1060A.

[00130] Cas proteins may contain at least one nuclease domain, such as a DNase domain. For example, the wild-type Cpf1 protein typically contains a RuvC-like domain that cleaves both strands of the target DNA, possibly in a dimeric configuration. Cas proteins may also contain at least two nuclease domains, such as DNase domains. For example, the wild-type Cas9 protein typically contains a RuvC-like nuclease domain and an HNH-like nuclease domain. Each of the RuvC and HNH domains can cut different strands of double stranded DNA to make a double strand break in the DNA. See, for example, Jinek et al. (2012) Science 337:816-821, incorporated herein by reference in its entirety for all purposes.

[00131] One or more nuclease domains may be deleted or mutated so that they no longer function or have reduced nuclease activity. For example, if one of the nuclease domains is deleted or mutated in the Cas9 protein, the resulting Cas9 protein can be called a nickase, and it can generate a nick in the target sequence for a guide RNA within a double-stranded DNA, but not a double-strand break (i.e., it can cleave complementary strand or non-complementary strand, but not both). An example of a mutation that converts Cas9 to nickase is the D10A (aspartate to alanine at position 10 in Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. Similarly, H939A (histidine to alanine at amino acid position 839), H840A (histidine to alanine at amino acid position 840), or N863 A (asparagine to alanine at amino acid position N863) in the HNH domain of Cas9 from S. pyogenes can convert Cas9 to nickase. Other examples of mutations that convert Cas9 to nickase include the corresponding mutations in Cas9 from S. thermophilics. See, for example, Sapranauskas et al. (2011) Nucleic Acids Research 39:9275-9282 and International Patent Publication WO 2013/141680, each document herein incorporated by reference in its entirety for all purposes. Such mutations can be generated using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or general gene synthesis. Examples of other nickase-creating mutations can be found, for example, in International Patent Publications WO 2013/176772 and WO 2013/142578, each of which is incorporated herein by reference in its entirety for all purposes.

[00132] Examples of inactivating mutations in the catalytic domains of Staphylococcus aureus Cas9 proteins are also known. For example, the Cas9 enzyme of Staphyloccocus aureus (SaCas9) may contain a substitution at position N580 (eg, substitution N580A) and a substitution at position D10 (eg, substitution D10A) to create a Cas protein with an inactive nuclease. See, for example, International Patent Publication WO 2016/106236, incorporated herein by reference in its entirety for all purposes.

[00133] Examples of inactivating mutations in the catalytic domains of Cpf1 proteins are also known. As for the Cpf1 proteins from U112 Francisella novicida (FnCpf1), BV3L6 Acidaminococcus sp. (AsCpf1), ND2006 Lachnospiraceae bacterium (LbCpf1) and 237 Moraxella bovoculi (MbCpf1 Cpf1), such mutations may include mutations at positions 908, 993 or 1263 in AsCpf1 or at the corresponding positions in the Cpf1 or 832, 925, 947 or 1180 orthologues in LbCpf1 or corresponding positions in Cpf1 orthologs. Such mutations may include, for example, one or more of D908A, E993A, and D1263A mutations in AsCpf1 or corresponding mutations in Cpf1 orthologs, or D832A, E925A, D947A and D1180A in LbCpf1 or corresponding mutations in Cpf1 orthologs. See, for example, US 2016/0208243, incorporated herein by reference in its entirety for all purposes.

[00134] Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, a Cas protein can be fused to a cleavage domain or an epigenetic modification domain. See International Patent Publication WO 2014/089290, incorporated herein by reference in its entirety for all purposes. Cas proteins can also be fused to a heterologous polypeptide providing increased or decreased stability. The fusion domain or heterologous polypeptide may be located at the N-terminus, C-terminus, or within the Cas protein.

[00135] As one example, a Cas protein can be fused to one or more heterologous polypeptides that provide subcellular localization. Such heterologous polypeptides may include, for example, one or more nuclear localization signals (NLS) such as SV40 single NLS and/or alpha importin double NLS for nuclear targeting, mitochondrial localization signal for mitochondrial targeting, ER retention signal etc. See, for example, Lange et al. (2007) J. Biol. Chem. 282:5101-5105, incorporated herein by reference in its entirety for all purposes. Such subcellular localization signals can be localized at the N-terminus, C-terminus, or anywhere on the Cas protein. The NLS may comprise a stretch of basic amino acids and may be a single sequence or a double sequence. Optionally, the Cas protein may contain two or more NLSs, including an NLS (eg, alpha import NLS or single NLS) at the N-terminus and an NLS (eg, SV40 NLS or double NLS) at the C-terminus. The Cas protein may also contain two or more NLSs at the N-terminus and/or two or more NLSs at the C-terminus.

[00136] Cas proteins can also be operably linked to a cell-penetrating or protein transduction domain. For example, the cell-penetrating domain can be derived from the HIV-1 TAT protein, the human hepatitis B virus TLM cell-penetrating motif, MPG, Pep-1, VP22, the cell-penetrating herpes simplex virus peptide, or the polyarginine peptide sequence. See, for example, International Patent Publications WO 2014/089290 and WO 2013/176772, each of which is incorporated herein by reference in its entirety for all purposes. The cell-penetrating domain can be located at the N-terminus, C-terminus, or anywhere on the Cas protein.

[00137] Cas proteins can also be operably linked to a heterologous polypeptide for ease of tracking or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, emerald green, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g. eBFP, eBFP2, azurite, mKalamal, GFPuv, sapphire, T-sapphire), blue fluorescent proteins (e.g. eCFP, sky blue, CyPet, AmCyanl, Midorii), red fluorescent proteins (e.g. mKate, mKate2, mPlum, DsRed-monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRespberry, mStrawberry, Jred), orange fluorescent proteins (eg mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein. Examples of labels include glutathione S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), nonn(NANP), tandem affinity purification label (TAP), myc, AcV5, AU1, AU5 , E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, histidine (His) , biotin carboxyl carrier protein (BCCP) and calmodulin.

[00138] A nucleic acid encoding a Cas protein can be codon optimized for efficient translation into a protein in a particular cell or organism. For example, a nucleic acid encoding a Cas protein can be modified to replace codons having a higher frequency of use in a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest according to compared to a naturally occurring polynucleotide sequence.

(3) Guide RNA

[00139] A "guide RNA" or "gRNA" is an RNA molecule that binds to a Cas protein (eg, the Cas9 protein) and targets the Cas protein to a specific location within the target DNA. Guide RNAs may contain two segments: a "DNA-targeting segment" and a "protein-binding segment". "Segment" includes a section or region of a molecule, such as a contiguous stretch of nucleotides in RNA. Some gRNAs, such as the Cas9 gRNA, may contain two separate RNA molecules: an "activator RNA" (eg, tracrPHK) and a "targeter RNA" (eg, CRISPR RNA or srRNA). Other gRNAs are a single RNA molecule (single RNA polynucleotide), which may also be referred to as "single molecule gRNA", "single guide RNA", or "sgRNA". See, for example, International Patent Publications WO 2013/176772, WO 2014/065596, WO 2014/089290, WO 2014/093622, WO 2014/099750, WO 2013/142578 and WO 2014/131833, each of which is incorporated herein in its entirety. document by reference for all purposes. For Cas9, for example, a single guide RNA may contain srRNA fused to tracrPHK (eg, via a linker). For Cpf1, for example, only srRNA is needed to achieve binding and/or cleavage of the target sequence. The terms "guide RNA" and "gRNA" include both two-molecule (ie, modular) gRNAs and single-molecule gRNAs.

[00140] A typical two-molecule gRNA contains a srRNA-like molecule ("CRISPR RNA", or "targeter-RNA", or "srRNA", or "srRNA repeat"), and a corresponding tracrPHK-like ("trans-active CRISPR RNA" or "activator-RNA" or "tracrPHK") molecule. srRNA contains both a DNA-targeting (single-stranded) gRNA segment and a stretch of nucleotides (ie, srRNA tail) that forms one half of the dsRNA duplex of the protein-binding gRNA segment. An exemplary srRNA tail downstream (3') of the DNA targeting segment contains, consists essentially of, or consists of GUUUUAGAGCUAUGCU (SEQ ID NO: 25). Any of the DNA targeting segments disclosed herein can be attached to the 5' end of SEQ ID NO: 25 to form srRNA.

[00141] The corresponding tracrRNA (activator-RNA) contains a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding gRNA segment. The nucleotide region of srRNA is complementary and hybridizes with the nucleotide region of tracrRNA to form a dsRNA duplex of the gRNA protein-binding domain. Thus, it can be said that each srRNA has a corresponding tracrRNA. An exemplary tracrPHK sequence contains, consists essentially of, or consists of AGCAUAGCAAGUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGCUUU (SEQ ID NO: 26).

[00142] In systems where both srRNA and tracrRNA are required, the srRNA and the corresponding tracrRNA hybridize to form gRNA. In systems that only require srRNA, the srRNA may be gRNA. In addition, srRNA provides a single-stranded DNA targeting segment that targets the guide RNA target sequence by hybridization to the opposite strand (ie, complementary strand). When used for intracellular modification, the exact sequence of a given srRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used. See, for example, Mali et al. (2013) Science 339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al. (2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol. 31:233-239; and Cong et al. (2013) Science 339:819-823, each of which is hereby incorporated by reference in its entirety for all purposes.

[00143] The DNA targeting segment (crRNA) of this gRNA contains a nucleotide sequence that is complementary to the sequence (i.e., the complementary strand of the guide RNA recognition sequence on the opposite strand of the target sequence for the guide RNA) in the target DNA. The DNA-targeting gRNA segment interacts with the target DNA in a sequence-specific manner through hybridization (ie, base pairing). As such, the nucleotide sequence of the DNA targeting segment can vary and determine the location within the target DNA at which the gRNA and target DNA will interact. The DNA targeting segment of the gRNA in question can be modified to hybridize to any desired sequence in the target DNA. Naturally occurring srRNAs vary by CRISPR/Cas system and organism, but often contain a targeting segment of 21 to 72 nucleotides in length flanked by two direct repeats (DR) of 21 to 46 nucleotides in length (see, e.g., International Patent Publication WO 2014/131833, incorporated herein by reference in its entirety for all purposes). In the case of S. pyogenes, DRs are 36 nucleotides long and the targeting segment is 30 nucleotides. The 3' DR is complementary and hybridizes to the corresponding tracrRNA, which in turn binds to the Cas protein.

[00144] The DNA targeting segment can be at least about 12 nucleotides long, at least about 15 nucleotides long, at least about 17 nucleotides long, at least about 18 nucleotides long, at least about 19 nucleotides long, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, or at least about 40 nucleotides. Such DNA targeting segments can be from about 12 nucleotides to about 100 nucleotides in length, from about 12 nucleotides to about 80 nucleotides, from about 12 nucleotides to about 50 nucleotides, from about 12 nucleotides to about 40 nucleotides, from about 12 nucleotides to about 30 nucleotides, from about 12 nucleotides to about 25 nucleotides, or from about 12 nucleotides to about 20 nucleotides. For example, a DNA targeting segment can be from about 15 nucleotides to about 25 nucleotides (e.g., from about 17 nucleotides to about 20 nucleotides, or about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, or about 20 nucleotides). See, for example, US 2016/0024523, incorporated herein by reference in its entirety for all purposes. For Cas9 from S. pyogenes, a typical DNA targeting segment is 16-20 nucleotides in length or 17-20 nucleotides in length. For Cas9 from S. aureus, a typical DNA targeting segment is 21-23 nucleotides in length. For Cpf1, a typical DNA targeting segment is at least 16 nucleotides in length, or at least 18 nucleotides in length.

[00145] tracrPHKs may be in any form (eg, full-length tracrPHKs or active partial tracrPHKs) and may be of any length. They include primary transcripts or processed forms. For example, tracrPHK (as part of a single guide RNA or as a single molecule as part of a bimolecular gRNA) may contain, consist essentially of, or consist of all or part of the wild-type tracrRNA sequence (e.g., about or greater than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracrRNA sequence). Exemplary wild-type tracrRNA sequences from S. pyogenes include 171 bp, 89 bp, 75 bp and 65 bp versions. See, for example, Deltcheva et al. (2011) Nature 471:602-607; International Patent Publication WO 2014/093661, each of which is incorporated herein by reference in its entirety for all purposes. Examples of tracrPHKs within single guide RNAs (sgPHKs) include the tracrPHK segments found within the +48, +54, +67, and +85 versions of sgPHK, where "+n" indicates that up to the +n nucleotide of wild-type tracrPHK type is included in sgPHK. See US Pat. No. 8,697,359, incorporated herein by reference in its entirety for all purposes.

[00146] The percentage of complementarity between the DNA targeting sequence and the complementary strand of the guide RNA recognition sequence within the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%), at least 90%, at least 95%, at least 97%, at least 98%), at least 99% or 100%). The percent complementarity between the DNA targeting sequence and the complementary strand of the guide RNA recognition sequence within the target DNA may be at least 60% over approximately 20 contiguous nucleotides. As an example, the percent complementarity between the DNA targeting sequence and the complementary strand of the guide RNA recognition sequence within the target DNA is 100% over 14 contiguous nucleotides at the 5' end of the complementary strand of the guide RNA recognition sequence within the complementary strand of the target DNA and only 0% over the rest. In such a case, the DNA targeting sequence can be considered to be 14 nucleotides in length. As another example, the percent complementarity between the DNA targeting sequence and the complementary strand of the guide RNA recognition sequence within the target DNA is 100% over seven contiguous nucleotides at the 5' end of the complementary strand of the guide RNA recognition sequence within the complementary strand of the target DNA, and only 0%. for the rest. In such a case, the DNA targeting sequence can be considered to be 7 nucleotides in length. In some guide RNAs, at least 17 nucleotides within the DNA targeting sequence are complementary to the target DNA. For example, a DNA targeting sequence may be 20 nucleotides in length and may contain 1, 2, or 3 mismatches to the complementary strand of the guide RNA recognition sequence. Optionally, mismatches are not adjacent to a protospacer adjacent motif (PAM) sequence (e.g., mismatches are at the 5' end of the DNA targeting sequence, or mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 base pairs from the PAM sequence).

[00147] A protein-binding gRNA segment may contain two stretches of nucleotides that are complementary to each other. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein-binding segment of the gRNA in question interacts with the Cas protein, and the gRNA directs the associated Cas protein to a specific nucleotide sequence within the target DNA via the DNA-targeting segment.

[00148] Single guide RNAs comprise a DNA targeting segment and a framework sequence (ie, a protein-binding or Cas-binding guide RNA sequence). For example, such guide RNAs contain a 5' DNA targeting segment and a 3' framework sequence. Exemplary framework sequences comprise, consist essentially of, or consist of the following: GUUUUAGAGCUAGAAAUAGCAAGUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCU (version 1; SEQ ID NO: 27); GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGC (version 2; SEQ ID NO: 6); GUUUUAGAGCUAGAAAUAGCAAGUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGC (version 3; SEQ ID NO: 7); and GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 4; SEQ ID NO: 8). Guide RNAs targeting any guide RNA target sequence may include, for example, a DNA targeting segment at the 5' end of the guide RNA fused to any of the exemplary guide RNA backbone sequences at the 3' end of the guide RNA. In other words, any of the DNA targeting segments disclosed herein can be joined to the 5' end of any of SEQ ID NO: 27, 6, 7 or 8 to form a single guide RNA (chimeric guide RNA). Versions 1, 2, 3, and 4 of the guide RNA, as disclosed elsewhere herein, are referred to as DNA targeting segments fused to scaffold versions 1, 2, 3, and 4, respectively.

[00149] Guide RNAs may include modifications or sequences that provide additional desired features (e.g., modified or adjusted stability; subcellular targeting; tracking with a fluorescent tag; binding site for a protein or protein complex; and the like). Examples of such modifications include, for example, the following: 5' cap (eg, 7-methylguanylate cap (m7G)); 3' polyadenylated tail (i.e. a 3' poly(A) tail); a switch RNA sequence (eg, to provide controlled stability and/or controlled availability of proteins and/or protein complexes); stability control sequence; a sequence that forms a dsRNA duplex (i.e. hairpin); a modification or sequence that directs the RNA to a subcellular location (eg, nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that allows tracking (eg, direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a binding site for proteins (eg, proteins that act on DNA, including DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like); and their combinations. Other examples of modifications include engineered loop-on-stem duplex structures, engineered bulge regions, engineered loop-on-stem duplex hairpins 3', or any combination thereof. See, for example, US 2015/0376586, incorporated herein by reference in its entirety for all purposes. The loopback may be an unpaired region of nucleotides in a duplex, consisting of a crRNA-like region and a minimal tracrRNA-like region. The loopback may contain, on one side of the duplex, an unpaired 5'-XXXY-3', where X is any purine and Y may be a nucleotide that can ambiguously pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex

[00150] Unmodified nucleic acids may be prone to degradation. Exogenous nucleic acids can also induce an innate immune response. Modifications can help introduce stability and reduce immunogenicity. Guide RNAs may contain modified nucleosides and modified nucleotides, including, for example, one or more of the following: (1) altering or replacing one or both of the non-binding phosphate oxygens and/or one or more binding phosphate oxygens in the backbone phosphodiester bond; (2) changing or replacing the ribose sugar component, for example, changing or replacing the 2' hydroxyl on the ribose sugar; (3) replacement of the phosphate moiety with dephospholinkers; (4) modification or replacement of a naturally occurring nucleic base; (5) replacement or modification of the ribose phosphate backbone; (6) modification of the 3' end or 5' end of the oligonucleotide (eg, removal, modification or replacement of the terminal phosphate group or conjugation of the fragment); and (7) sugar modification. Other possible modifications to the guide RNA include modifications or substitutions of uracils or polyuracil pathways. See, for example, International Patent Publication WO 2015/048577 and US 2016/0237455, each of which is incorporated herein by reference in its entirety for all purposes. Similar modifications can be made to Cas-encoding nucleic acids, such as Cas mRNA.

[00151] As one example, the nucleotides at the 5' or 3' end of the guide RNA may include phosphorothioate linkages (eg, bases may contain a modified phosphate group that is a phosphorothioate group). For example, the guide RNA may include phosphorothioate bonds between the 2, 3 or 4 terminal nucleotides at the 5' or 3' end of the guide RNA. As another example, the nucleotides at the 5' and/or 3' end of the guide RNA may contain 2'-O-methyl modifications. For example, the guide RNA may include 2'-O-methyl modifications at the 2, 3, or 4 terminal nucleotides at the 5' and/or 3' end of the guide RNA (eg, at the 5' end). See, for example, WO 2017/173054 A1 and Finn et al. (2018) Cell Reports 22:1-9, each of which is hereby incorporated by reference in its entirety for all purposes. In one specific example, the guide RNA contains 2'-O-methyl analogs and 3'-phosphorothioate internucleotide bonds at the first three 5' and 3' terminal residues of the RNA. In another specific example, the guide RNA is modified so that all 2'OH groups that do not interact with the Cas9 protein are replaced with 2'-O-methyl analogs, and the tail region of the guide RNA, which has minimal interaction with Cas9, is modified with 5' and 3' phosphorothioate internucleotide bonds. See, for example, Yin et al. (2017) Nat. Biotech. 3 5 (12): 1179-1187, incorporated herein by reference in its entirety for all purposes.

[00152] Guide RNAs can be provided in any form. For example, the gRNA can be provided in the form of an RNA, either as two molecules (single srRNA and tracrPHK) or as a single molecule (sgRNA) and optionally in the form of a complex with a Cas protein. The gRNA may also be provided in the form of DNA encoding the gRNA. The DNA encoding gRNA may encode a single RNA molecule (sgRNA) or individual RNA molecules (eg, individual srRNA and tracrRNA). In the latter case, the DNA encoding the gRNA may be provided as a single DNA molecule or as separate DNA molecules encoding srRNA and tracrPHK, respectively.

[00153] When the gRNA is provided in the form of DNA, the gRNA may be transiently, conditionally, or constitutively expressed in the cell. DNA encoding gRNA can be stably integrated into the cell genome and functionally linked to an active promoter in the cell. Alternatively, the DNA encoding the gRNA may be operably linked to a promoter in an expression construct. For example, the DNA encoding the gRNA may be in a vector containing a heterologous nucleic acid, such as a nucleic acid encoding a Cas protein. Alternatively, it may be a vector or a plasmid that is separated from a vector containing a nucleic acid encoding a Cas protein. Promoters that can be used in such expression constructs include promoters active in, for example, one or more of the following: eukaryotic cell, human cell, non-human cell, mammalian cell, non-human mammalian cell, rodent cell , rat cell, hamster cell, rabbit cell, pluripotent cell, embryonic stem (ES) cell, adult human stem cell, developmentally restricted progenitor cell, induced pluripotent stem cell (iPS), or embryo at the single cell stage. Such promoters may be, for example, conditional promoters, inducible promoters, constitutive promoters or tissue-specific promoters. Such promoters can also be, for example, bidirectional promoters. Specific examples of suitable promoters include an RNA polymerase III promoter such as the human U6 promoter, the rat U6 polymerase III promoter, or the mouse U6 polymerase III promoter.

[00154] Alternatively, gRNA can be obtained in various other ways. For example, gRNA can be produced by in vitro transcription using, for example, T7 RNA polymerase (see, for example, International Patent Publications WO 2014/089290 and WO 2014/065596, each of which is incorporated herein by reference in its entirety for all purposes). ). Guide RNAs can also be a synthetically produced molecule obtained by chemical synthesis.

(4) Guide RNA recognition sequences and guide RNA target sequences

[00155] The term "guide RNA recognition sequence" includes nucleic acid sequences present in a target DNA that will bind to a DNA-targeting gRNA segment, provided sufficient binding conditions are present. As used herein, the term "guide RNA recognition sequence" encompasses both strands of the double-stranded target DNA (i.e., the sequence on the complementary strand to which the guide RNA hybridizes and the corresponding sequence on the non-complementary strand adjacent to the protospacer adjacent motif (PAM) ). As used herein, the term "target sequence for a guide RNA" refers specifically to a sequence on a non-complementary strand adjacent to the PAM (ie, upstream or 5' from the PAM). That is, the target sequence for the guide RNA refers to the sequence on the non-complementary strand corresponding to the sequence to which the guide RNA hybridizes on the complementary strand. The target sequence for the guide RNA is equivalent to the DNA targeting segment of the guide RNA, but with thymines instead of uracils. As one example, the target sequence for a guide RNA for the Cas9 enzyme may refer to the sequence on the non-complementary strand adjacent to the 5'-NGG-3' PAM. Guide RNA recognition sequences include sequences to which the guide RNA is intended to provide complementarity, wherein hybridization between the complementary strand of the guide RNA recognition sequence and the DNA targeting guide RNA sequence promotes the formation of a CRISPR complex. Complete complementarity is not necessarily necessary, provided that sufficient complementarity exists to induce hybridization and facilitate the formation of the CRISPR complex. Guide RNA recognition sequences or guide RNA target sequences also include cleavage sites for Cas proteins, described in more detail below. The guide RNA recognition sequence or guide RNA target sequences may comprise any polynucleotide, which may be located, for example, in the nucleus or cytoplasm of a cell, or in a cell organelle such as a mitochondrion or chloroplast.

[00156] The guide RNA recognition sequence in the target DNA can be targeted (i.e., linked to, or can hybridize to, or be complementary to) a Cas protein or gRNA. Suitable DNA/RNA binding conditions include the physiological conditions normally found in the cell. Other suitable DNA/RNA binding conditions (e.g., conditions in a cell-free system) are known (see, e.g., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), incorporated in its entirety in this document by reference for any purpose). A target DNA strand that is complementary and hybridizes to a Cas protein or gRNA can be called a "complementary strand" and a target DNA strand that is complementary to the "complementary strand" (and therefore not complementary to a Cas protein or gRNA) can be called a "non-complementary strand" or "matrix chain".

[00157] The Cas protein can cleave the nucleic acid at a site within or outside the nucleic acid sequence present in the target DNA to which the DNA-targeting gRNA segment will bind. "Cleavage site" includes the position of the nucleic acid at which the Cas protein causes a single-strand break or a double-strand break. For example, formation of a CRISPR complex (comprising a gRNA hybridized to the complementary strand of the guide RNA recognition sequence and complexed with the Cas protein) can result in cleavage of one or both strands at or near (e.g., within 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 20, 50 or more base pairs from) a nucleic acid sequence present in the target DNA that will bind to the DNA targeting gRNA segment. If the cleavage site is outside the nucleic acid sequence to which the DNA-targeting gRNA segment will bind, the cleavage site is still considered to be in the "guide RNA recognition sequence" or guide RNA target sequence. The cleavage site may be on only one strand or on both strands of the nucleic acid. The cleavage sites may be at the same position on both strands of the nucleic acid (creating blunt ends) or may be at different sites on each strand (creating staggered ends (ie, hanging ends)). Staggered ends can be generated, for example, using two Cas proteins, each causing a single strand break at a different cleavage site on the other strand, thereby creating a double strand break. For example, the first nickase can create a single strand break on the first strand of double stranded DNA (dsDNA), and the second nickase can create a single strand break on the second strand of dsDNA so that dangling sequences are created. In some cases, the guide RNA recognition sequence or target sequence for nickase guide RNA on the first strand is separated from the guide RNA recognition sequence or target sequence for nickase guide RNA on the second strand by at least 2, 3, 4 years. 5, 6, 7, 8 , 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500 or 1000 bp.

[00158] Site-specific binding and/or cleavage of the target DNA by Cas proteins can occur at locations defined by both (i) base pairing complementarity between gRNA and target DNA, and (ii) a short motif called protospacer adjacent motif (PAM ) in the target DNA. The PAM can flank the target sequence for the guide RNA on a non-complementary strand opposite the strand to which the guide RNA hybridizes. Optionally, the target sequence for the guide RNA may be flanked at the 3' end of the PAM. Alternatively, the target sequence for the guide RNA can be flanked at the 5' end of the PAM. For example, the cleavage site of Cas proteins can be about 1 to about 10, or about 2 to about 5 base pairs (eg, 3 base pairs) upstream or downstream of the PAM sequence. In some cases (for example, when Cas9 from S. pyogenes or a closely related Cas9 is used), the non-complementary strand PAM sequence may be 5'-NiGG-3', where Ni is any DNA nucleotide and is located immediately 3' from the non-complementary guide RNA recognition sequence. target DNA strand (i.e. immediately 3' from the target sequence for the guide RNA). Thus, the PAM sequence of the complementary strand will be 5'-CCN2-3', where N ₂ is any DNA nucleotide and is immediately 5' from the guide RNA recognition sequence of the complementary strand of the target DNA. In some such cases, N ₁ and N ₂ may be complementary, and the base pair N ₁ -N ₂ may be any base pair (for example, N ₁ =C and N ₂ =G; N ₁ =G and N ₂ =C; N ₁ =A and N ₂ =T; or N ₁ =T and N ₂ =A). In the case of Cas9 from S. aureus, the PAM may be NNGRRT or NNGRR, where N may be A, G, C, or T, and R may be G or A. In the case of Cas9 from C. jejuni, PAM may be, for example , NNNNACAC or NNNNRYAC, where N may be A, G, C, or T and R may be G or A. In some cases (for example, for FnCpf1), the PAM sequence may be upstream from the 5' end and be characterized by the sequence 5'-TTN-3'.

[00159] Examples of guide RNA target sequences or guide RNA target sequences in addition to the PAM sequence are provided below. For example, the target sequence for a guide RNA may be the 20 nucleotide DNA sequence immediately preceding the NGG motif recognized by the Cas9 protein. Examples of such target guide RNA sequences together with the PAM sequence are GN ₁₉ NGG (SEQ ID NO: 9) or N ₂₀ NGG (SEQ ID NO: 10). See, for example, International Patent Publication WO 2014/165825 incorporated by reference in its entirety for all purposes. Guanine at the 5' end can facilitate transcription by RNA polymerase in cells. Other examples of target sequences for the guide RNA along with the PAM sequence may include two guanine nucleotides at the 5' end (eg, GGN ₂₀ NGG; SEQ ID NO: 11) to facilitate efficient transcription by T7 polymerase in vitro. See, for example, International Patent Publication WO 2014/065596 incorporated by reference in its entirety for all purposes. Other target sequences for the guide RNA together with the PAM sequence may be 4 to 22 nucleotides in length according to SEQ ID NOs: 9-11, including 5' G or GG and 3' GG or NGG. Another target sequence for the guide RNA may be 14 to 20 nucleotides in length according to SEQ ID NOs: 9-11.

[00160] The guide RNA recognition sequence or guide RNA target sequence can be any nucleic acid sequence endogenous or exogenous to the cell. The guide RNA recognition sequence or guide RNA target sequence may be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory sequence), or may include both.

B. Cells and Non-Human Animals Containing Cas Expression Cassettes

[00161] Also provided are cells and non-human animals containing the Cas expression cassettes described herein. The Cas expression cassette may be stably integrated into the genome (ie, chromosome) of a cell or non-human animal, or it may be located outside the chromosome (eg, extrachromosomally replicating DNA). Optionally, the Cas expression cassette is stably integrated into the genome. A stably integrated Cas expression cassette may be randomly integrated into the genome of a non-human (ie, transgenic) animal, or it may be integrated into a predetermined region of the genome of a non-human (ie, knockin) animal. Optionally, the Cas expression cassette stably integrates into a predetermined region of the genome, such as a safe harbor locus. The target genomic locus at which the Cas expression cassette is stably integrated can be heterozygous for the Cas expression cassette or homozygous for the Cas expression cassette. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a particular genetic locus. Genotypes are described as homozygous if there are identical alleles present at a particular locus, and as heterozygous if two alleles are different.

[00162] The cells provided herein may be, for example, eukaryotic cells, which include, for example, fungal (e.g., yeast), plant cells, animal cells, mammalian cells, non-human mammalian cells, and human cells. The term "animal" includes mammals, fish and birds. The mammalian cell can be, for example, a non-human mammalian cell, a human cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates, apes, great apes, cats, dogs, rabbits, horses, oxen, deer, bison, livestock (for example, cattle species such as cows, bulls, etc., ovine species such as sheep, goats, etc., and swine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, and so on. Pets and farm animals are also provided. The term "non-human" excludes humans.

[00163] Cells can also be in an undifferentiated or differentiated state of any type. For example, the cell may be a totipotent cell, a pluripotent cell (eg, a human pluripotent cell or a non-human pluripotent cell such as a mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotent cell. Totipotent cells include undifferentiated cells that can give rise to any type of cell, and pluripotent cells include undifferentiated cells that have the ability to develop into more than one type of differentiated cell. Such pluripotent and/or totipotent cells may be, for example, ES cells or ES-like cells such as induced pluripotent stem (iPS) cells. ES cells include embryonic-derived totipotent or pluripotent cells that are capable of contributing to any tissue in the developing embryo when introduced into the embryo. ES cells can be derived from the inner cell mass of the blastocyst and are capable of differentiating into cells from any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[00164] Examples of human pluripotent cells include human ES cells, human adult stem cells, developmentally restricted human progenitor cells, and human induced pluripotent stem (iPS) cells such as primed human iPS cells and naïve human iPS cells. Induced pluripotent stem cells include pluripotent stem cells that can be obtained directly from a differentiated adult cell. Human iPS cells can be prepared by introducing specific sets of reprogramming factors into the cell, which may include, for example, Oct3/4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Mus, 1-Mus, p-Mus), Krüppel-like family (KLF) transcription factors (eg KLF1, KLF2, KLF4, KLF5) and/or related transcription factors such as NANOG, LIN28 and/or Glis1. Human iPS cells can also be generated, for example, by using microRNAs, small molecules that mimic the action of transcription factors or lineage specifiers. Human iPS cells are characterized by their ability to differentiate into any of the three germ layers of vertebrates, such as endoderm, ectoderm, or mesoderm. Human iPS cells are also characterized by their ability to proliferate indefinitely under suitable in vitro culture conditions. See, for example, Takahashi and Yamanaka (2006) Cell 126: 663-676, incorporated herein by reference in its entirety for all purposes. Human ES-primed cells and human iPS-primed cells include cells that exhibit characteristics similar to those of epiblast cells after implantation and are intended for lineage specification and differentiation. Naïve human ES cells and naïve human iPS cells include cells that exhibit characteristics similar to those of ES cells of the inner cell mass of a preimplantation embryo and are not required for lineage characterization. See, for example, Nichols and Smith (2009) Cell Stem Cell 4:487-492, incorporated herein by reference in its entirety for all purposes.

[00165] The cells provided herein may also be germ cells (eg, spermatozoa or oocytes). The cells may be mitotically competent cells or mitotically inactive cells, meiotically competent cells or meiotically inactive cells. Similarly, the cells may also be primary somatic cells or cells that are not primary somatic cells. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the cells may be liver cells, kidney cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, blood cells, melanocytes, monocytes, mononuclear cells, monocytic progenitors, B cells, erythroid megakaryocytic cells, eosinophils , macrophages, T cells, islet beta cells, exocrine cells, pancreatic progenitors, endocrine progenitors, adipocytes, preadipocytes, neurons, glial cells, neural stem cells, neurons, hepatoblasts, hepatocytes, cardiomyocytes, skeletal myoblasts, smooth muscle cells, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, cholangiocytes, white or brown adipocytes, or eye cells (eg, trabecular meshwork cells, retinal pigment epithelial cells, retinal microvascular endothelial cells, retinal pericytes, conjunctival epithelial cells, conjunctival fibroblasts, epithelial cells and iris pigment, keratocytes, lens epithelial cells, non-pigmented ciliary epithelial cells, ocular choroid fibroblasts, photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, or amacrine cells).

[00166] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell derived from an organism, organ, or tissue that has not previously been transferred to tissue culture, or has previously been transferred to tissue culture, but is not capable of being transferred to tissue culture indefinitely. Such cells can be isolated using conventional techniques and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes, neurons, glial cells. , hepatocytes, skeletal myoblasts and smooth muscle cells. For example, primary cells may be derived from connective tissues, muscle tissues, nervous system tissues, or epithelial tissues.

[00167] Other suitable cells provided herein include immortalized cells. Immortalized cells are cells of a multicellular organism that do not normally reproduce indefinitely, but due to mutation or change have escaped normal cellular aging and may instead continue to undergo division. Such mutations or changes may occur naturally or be intentionally induced. Examples of immortalized cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (eg, HEK 293 cells or 293T cells), and mouse embryonic fibroblasts (eg, 3T3 cells). Numerous types of immortalized cells are well known. Immortal or primary cells include cells that are typically used for culture or for the expression of recombinant genes or proteins.

[00168] The cells provided herein also include embryos at the single cell stage (i.e., fertilized oocytes or zygotes). Such single cell embryos may be from any genetic background (eg, BALB/c, C57BL/6, 129, or combinations thereof in mice), may be fresh or frozen, and may be derived from natural breeding or in vitro fertilization.

[00169] The cells provided herein may be normal, healthy cells, or may be pathological or mutated cells.

[00170] Non-human animals containing a Cas expression cassette as described herein can be produced using the methods described elsewhere herein. The term "animal" includes mammals, fish and birds. Mammals include, for example, humans, non-human primates, apes, great apes, cats, dogs, rabbits, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs). ) and livestock (for example, bovine species such as cows and bulls; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese and ducks. Pets and farm animals are also provided. The term "non-human" excludes humans. Preferred non-human animals include, for example, rodents such as mice and rats.

[00171] Non-human animals can be of any genetic origin. For example, suitable mice may be the 129 line, the C57BL/6 line, a mixture of 129 and C57BL/6, the BALB/c line, or the Swiss Webster line. Examples of lines 129 include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac, 129S7), and 129T2. See, for example, Festing et al. (1999) Mammalian Genome 10:836, incorporated herein by reference in its entirety for all purposes. Examples of C57BL lineages include C57BL/A, C57BL/An, C57BL/GrFa, C57BL/Kal wN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/01a. Suitable mice can also be from a mixture of the aforementioned 129 line and the aforementioned C57BL/6 line (eg 50% 129 and 50% C57BL/6). Likewise, suitable mice can be from a mixture of the aforementioned 129 lines or a mixture of the aforementioned BL/6 lines (eg, the 129S6 (129/SvEvTac) line).

[00172] Similarly, the rats can be from any strain of rats, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fischer like Fisher F344 or Fisher F6. Rats can also be obtained from a line derived from a mixture of two or more of the lines above. For example, a suitable rat may be from the DA strain or the ACI strain. The ACI rat strain is characterized by having a black agouti coat color, white belly and paws, and the RT1 ^av1 haplotype. Such lines are available from various sources, including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized by having an agouti coat color and the RT1 ^av1 haplotype. Such rats are available from a variety of sources including Charles River and Harlan Laboratories. In some cases, suitable rats may be inbred rats. See, for example, US 2014/0235933, which is hereby incorporated by reference in its entirety for all purposes.

C. Target genomic loci

[00173] The Cas expression cassettes described herein may be genomically integrated at a target genomic locus in a non-human animal cell or body. Any target genomic locus capable of expressing the gene can be used.

[00174] An example of a target genomic locus into which the Cas expression cassettes described herein can stably integrate is a safe harbor locus in the genome of a non-human animal. Interactions between integrated exogenous DNA and the host genome may limit the reliability and safety of integration and may result in overt phenotypic effects that are not due to the targeted genetic modification but instead are due to unintended effects of integration on surrounding endogenous genes. For example, randomly inserted transgenes can be subject to positional effects and silencing, making their expression unreliable and unpredictable. Similarly, integration of exogenous DNA into a chromosomal locus can influence surrounding endogenous genes and chromatin, thereby altering cell behavior and phenotypes. Safe harbor loci include chromosomal loci where transgenes or other exogenous nucleic acid inserts can be stably and reliably expressed in all tissues of interest without overt alteration of cell behavior or phenotype (i.e. without any detrimental effects on the cell- owner). See, for example, Sadelain et al. (2012) Nat. Rev. Cancer 12:51-58, incorporated herein by reference in its entirety for all purposes. Optionally, a safe harbor locus is one at which the expression of the inserted gene sequence is not disturbed by any pass-through expression from neighboring genes. For example, safe harbor loci may include chromosomal loci where exogenous DNA can integrate and function in a predictable manner without adversely affecting the structure or expression of endogenous genes. Safe harbor loci may include extragenic regions or intragenic regions, such as, for example, loci in genes that are unnecessary, optional, or can be disrupted without obvious phenotypic consequences.

[00175] For example, the Rosa26 locus and its human equivalent provide an open chromatin configuration in all tissues and is ubiquitously expressed during embryonic development and in adults. See, for example, Zambrowicz et al. (1997) Proc. Natl. Acad. sci. USA 94:3789-3794, incorporated herein by reference in its entirety for all purposes. In addition, the Rosa26 locus can be targeted with high efficiency, and disruption of the Rosa26 gene does not result in a clear phenotype. Other examples of safe harbor loci include CCR5, HPRT, AAVS1, and albumin. See, for example, US Pat. Nos. 7,888,121; 7972854; 7914796; 7951925; 8110379; 8409861; 8586526; and US Patent Publication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2006/0063231; 2008/0159996; 2010/00218264; 2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591; 2013/0177983; 2013/0177960 and 2013/0122591, each document hereby incorporated by reference in its entirety for all purposes. Biallelic targeting of safe harbor loci such as the Rosa26 locus has no negative consequences, so different genes or reporters can target two Rosa26 alleles. In one example, the Cas expression cassette is integrated into an intron of the Rosa26 locus, such as the first intron of the Rosa26 locus.

D. Non-Human Animals In Which Recombinase Cuts a DNA Section

[00176] Cells or non-human animals containing a Cas expression cassette containing a Cas coding sequence downstream of a polyadenylation signal or a transcription terminator flanked by recombinase recognition sites recognized by a site-specific recombinase as disclosed herein may further contain an expression a recombinase cassette that directs the expression of a site-specific recombinase. Site-specific recombinases include enzymes that can promote recombination between recombinase recognition sites where two recombination sites are physically separated in the same nucleic acid or in separate nucleic acids. Examples of recombinases include Cre, Flp and Dre recombinases. One example of a Cre recombinase gene is Crei, in which two exons encoding Cre recombinase are separated by an intron to prevent its expression in a prokaryotic cell. Such recombinases may additionally contain a nuclear localization signal to facilitate nuclear localization (eg, NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by a site-specific recombinase and can serve as a substrate for a recombination event. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2 and lox5171.

[00177] The Cas expression cassette and the recombinase expression cassette may be integrated into different target genomic loci, or they may be genomically integrated into the same target locus (e.g., the Rosa26 locus, for example, integrated into the first intron of the Rosa26 locus). For example, a cell or non-human animal may be heterozygous for each of a Cas expression cassette and a recombinase expression cassette, with one allele of the target genomic locus containing the Cas expression cassette and a second allele of the target genomic locus containing the recombinase expression cassette.

[00178] The recombinase gene in the recombinase expression cassette can be operably linked to any suitable promoter. Examples of promoters are disclosed elsewhere in this document. For example, the promoter may be a tissue-specific promoter (eg, an albumin promoter for liver-specific expression or an insulin 2 promoter for pancreatic-specific expression) or a developmental stage-specific promoter. An advantage provided by such promoters is that Cas expression can be activated selectively in the desired tissue or only at the desired developmental stage, thereby reducing the possibility of Cas-mediated toxicity in vivo. A non-limiting list of illustrative promoters for mouse strains in which DNA excision is performed by recombinase is presented in Table 2.

[00179]

III. Methods for assessing CRISPR/Cas activity in vivo

[00180] Various methods are provided for evaluating delivery of CRISPR/Cas to tissues and organs of a living animal and evaluating CRISPR/Cas activity in tissues and organs of a living animal. Such methods use non-human animals containing a Cas expression cassette as described elsewhere herein.

A. Methods for testing the ability of CRISPR/Cas to modify a target genomic locus in vivo or ex vivo

[00181] Various methods are provided to assess the ability of a nickase or a CRISPR/Cas nuclease to modify a target genomic locus in vivo using non-human animals containing a Cas expression cassette as described herein. Such methods may include: (a) administering to a non-human animal a guide RNA designed to target a guide RNA target sequence at a target genomic locus; and (b) assessing the modification of the target locus. Modification of the target genomic locus will be induced when the guide RNA forms a complex with the Cas protein and directs the Cas protein to the target genomic locus, and the Cas/guide RNA complex cleaves the target sequence for the guide RNA, triggering cellular repair (e.g., via non-homologous end joining (NHEJ) if there is no donor sequence).

[00182] Optionally, two or more guide RNAs can be introduced, each designed to target a different target sequence for the guide RNA within the target genomic locus. For example, two guide RNAs may be designed to cut a genomic sequence between two target guide RNA sequences. Modification of the target genomic locus will be induced when the first guide RNA complex with the Cas protein and direct the Cas protein to the target genomic locus, the second guide RNA complex with the Cas protein and direct the Cas protein to the target genomic locus, the first Cas/guide RNA complex cleaves the first guide RNA target sequence, and the second Cas/guide RNA complex cleaves the second guide RNA target sequence, resulting in excision of the intermediate sequence. Alternatively, two or more guide RNAs can be introduced, each designed to target a different target sequence for the guide RNA at a different target genomic locus (ie multiplexing).

[00183] Optionally, an exogenous donor nucleic acid capable of recombination and modification of the target genomic locus is also administered to the non-human animal. Optionally, the Cas protein can be attached to an exogenous donor nucleic acid, as described elsewhere herein. Optionally, two or more exogenous donor nucleic acids are introduced, each of which is capable of recombination and modification of a different target genomic locus. Modification of the target genomic locus will be induced, for example, when the guide RNA forms a complex with the Cas protein and directs the Cas protein to the target genomic locus, the Cas/guide RNA complex cleaves the target sequence for the guide RNA, and the target genomic locus is recombined with the exogenous donor nucleic acid to modify the target genomic locus. The exogenous donor nucleic acid can be recombined with the target genomic locus, for example, via homologous repair (HDR) or via NHEJ-mediated insertion. You can use any type of exogenous donor nucleic acid, examples of which are provided elsewhere in this document.

[00184] Similarly, the various methods provided above for assessing in vivo CRISPR/Cas activity can also be used to assess ex vivo CRISPR/Cas activity using cells containing the Cas expression cassette, as described elsewhere herein.

[00185] Guide RNAs and optionally exogenous donor nucleic acids can be introduced into a cell or non-human animal via any delivery method (e.g., AAV, LNP, or HDD) and any route of administration as disclosed elsewhere herein. According to specific methods, the guide RNA (or guide RNAs) is delivered via AAV-mediated delivery. For example, AAV8 can be used if the liver is the target.

[00186] Methods for assessing modification of a target genomic locus are provided elsewhere herein and are well known. The target genomic locus modification assessment can be performed in any cell type, any tissue type, or any organ type, as disclosed elsewhere herein. In some methods, the modification of the target genomic locus is assessed in liver cells (eg, assessment of serum levels of secreted protein expressed by the target genomic locus in liver cells). For example, the target genomic locus contains the target gene, and the assessment may involve measuring the expression of the target gene or the activity of the protein encoded by the target gene. Alternatively or additionally, the evaluation may comprise sequencing the target genomic locus in one or more cells isolated from a non-human animal. The evaluation may comprise isolating the target organ or tissue from a non-human animal and assessing the modification of the target genomic locus in the target organ or tissue. The assessment may also comprise an assessment of the modification of the target genomic locus in two or more different cell types within the target organ or tissue. Similarly, the assessment may comprise isolating a non-target organ or tissue (eg, two or more non-target organs or tissues) from a non-human animal and assessing the modification of the target genomic locus in the non-target organ or tissue.

(1) Exogenous donor nucleic acids

[00187] The methods and compositions disclosed herein use exogenous donor nucleic acids to modify a target genomic locus after cleaving the target genomic locus with a Cas protein. In such methods, the Cas protein cleaves the target genomic locus to create a single-strand break (nick) or double-strand break, and the exogenous donor nucleic acid is recombined with the target genomic locus via non-homologous end joining (NHEJ) mediated ligation or through a homologous repair phenomenon. Optionally, repair with an exogenous donor nucleic acid removes or destroys the guide RNA target sequence or Cas cleavage site so that alleles that have been targeted cannot be retargeted by the Cas protein.

[00188] Exogenous donor nucleic acids may contain deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), they may be single-stranded or double-stranded, and they may be in linear or circular form. For example, the exogenous donor nucleic acid may be a single stranded oligodeoxynucleotide (ssODN). See, for example, Yoshimi et al. (2016) Nat. commun. 7:10431, incorporated herein by reference in its entirety for all purposes. An exemplary exogenous donor nucleic acid is from about 50 nucleotides to about 5 kb. in length, from about 50 nucleotides to about 3 kb. in length, or from about 50 to about 1000 nucleotides in length. Other illustrative exogenous donor nucleic acids are from about 40 to about 200 nucleotides in length. For example, exogenous donor nucleic acid may be approximately 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150 -160, 160-170, 170-180, 180-190 or 190-200 nucleotides in length. Alternatively, the exogenous donor nucleic acid may be approximately 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900 or 900-1000 nucleotides per length. Alternatively, the exogenous donor nucleic acid may be approximately 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5 or 4.5-5 kb. in length. Alternatively, the exogenous donor nucleic acid may be, for example, no more than 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, or 50 nucleotides in length. Exogenous donor nucleic acids (eg, targeting vectors) can also be longer.

[00189] According to one example, the exogenous donor nucleic acid is an ssODN that is from about 80 nucleotides to about 200 nucleotides in length. According to another example, exogenous donor nucleic acids are ssODN, which is from about 80 nucleotides to about 3 kb. in length. Such ssODN may contain homologous arms, for example, each of which is from about 40 nucleotides to about 60 nucleotides in length. Such ssODN may also contain homologous arms, for example, each of which is from about 30 nucleotides to 100 nucleotides in length. Homologous arms can be symmetrical (for example, each is 40 nucleotides long or each is 60 nucleotides long), or they can be asymmetrical (for example, one homologous arm that is 36 nucleotides long and one homologous arm that is 91 nucleotides long). length).

[00190] Exogenous donor nucleic acids may include modifications or sequences that provide additional desired features (e.g., altered or controlled stability; tracking or detection with a fluorescent label; binding site for a protein or protein complex, etc.). Exogenous donor nucleic acids may contain one or more fluorescent labels, purification labels, epitope labels, or a combination thereof. For example, the exogenous donor nucleic acid may contain one or more fluorescent labels (e.g., fluorescent proteins or other fluorophores or dyes), such as at least 1, at least 2, at least 3, at least 4, or at least 5 fluorescent labels. Exemplary fluorescent labels include fluorophores such as fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-(and-6)-carboxytetramethylrhodamine ( TAMRA) and Su7. A wide range of fluorescent dyes are commercially available for labeling oligonucleotides (eg from Integrated DNA Technologies). Such fluorescent labels (eg, internal fluorescent labels) can be used, for example, to detect exogenous donor nucleic acid that is directly integrated into a cleaved target nucleic acid containing overhangs that are compatible with the ends of the exogenous donor nucleic acid. The label or tag may be at the 5' end, 5' end, or within the exogenous donor nucleic acid. For example, an exogenous donor nucleic acid can be conjugated at the 5' end to Integrated DNA Technologies' IR700 fluorophore ( ^5'IRDYE® 700).

[00191] Exogenous donor nucleic acids may also contain nucleic acid inserts, including DNA segments to be integrated at target genomic loci. Integration of a nucleic acid insert at a target genomic locus may result in the addition of a nucleic acid sequence of interest at a target genomic locus, a deletion of a nucleic acid sequence of interest at a target genomic locus, or a substitution of a nucleic acid sequence of interest at a target genomic locus (i.e., deletion and insertion ). Some exogenous donor nucleic acids are designed to insert a nucleic acid insert at a target genomic locus without any corresponding deletion at the target genomic locus. Other exogenous donor nucleic acids of a nucleic acid sequence of interest to be deleted at a target genomic locus without any corresponding insertion of a nucleic acid insert. Other exogenous donor nucleic acids are designed to delete the nucleic acid sequence of interest at the target genomic locus and replace it with a nucleic acid insert.

[00192] The nucleic acid insert or corresponding nucleic acid at the target genomic locus to be deleted and/or replaced may be of varying length. An exemplary nucleic acid insert or corresponding nucleic acid at a target genomic locus to be deleted and/or replaced is from about 1 nucleotide to about 5 kb. in length, or from about 1 nucleotide to about 1000 nucleotides in length. For example, the nucleic acid insert or corresponding nucleic acid at the target genomic locus to be deleted and/or replaced may be approximately 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70 , 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190 or 190 -120 nucleotides in length. Similarly, the nucleic acid insert or corresponding nucleic acid at the target genomic locus to be deleted and/or replaced may be 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900 or 900-1000 nucleotides in length. Similarly, the nucleic acid insert or corresponding nucleic acid at the target genomic locus to be deleted and/or replaced may be approximately 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3 .5, 3.5-4, 4-4.5 or 4.5-5 kb. in length or more.

[00193] The nucleic acid insert may contain a sequence that is homologous or orthologous to all or part of the sequence targeted for replacement. For example, the nucleic acid insert may contain a sequence that contains one or more point mutations (eg, 1, 2, 3, 4, 5 or more) compared to the sequence targeted for replacement at the target genomic locus. Optionally, such point mutations may result in a conservative amino acid substitution (eg, substitution of aspartic acid [Asp, D] with glutamic acid [Glu, E]) in the encoded polypeptide.

(2) Donor Nucleic Acids for Nonhomologous End Joining Mediated Insertion

[00194] Some exogenous donor nucleic acids contain short single-stranded regions at the 5' end and/or 3' end that are complementary to one or more overhangs created by Cas-mediated cleavage at the target genomic locus. These hanging ends may also be referred to as 5' and 3' homologous arms. For example, some exogenous donor nucleic acids contain short single-stranded regions at the 5' end and/or 3' end that are complementary to one or more drooping ends created by Cas-mediated cleavage at the 5' and/or 3' target sequences in the target genomic locus. Some such exogenous donor nucleic acids contain a complementary region only at the 5' end or only at the 3' end. For example, some such exogenous donor nucleic acids contain a complementary region only at the 5' end, which is complementary to an overhanging end generated at the 5' end of the target sequence at the target genomic locus, or only at the 3' end, complementary to the overhanging end generated at the 3' end of the target sequence at the target genomic locus. genomic locus. Other such exogenous donor nucleic acids contain complementary regions at both the 5' and 3' ends. For example, other such exogenous donor nucleic acids contain complementary regions at both the 5' and 3' ends, eg, complementary to the first and second overhangs, respectively, created by Cas-mediated cleavage at the target genomic locus. For example, if the exogenous donor nucleic acid is double-stranded, single-stranded complementary regions may extend from the 5' end of the donor nucleic acid's upper strand and the 5' end of the donor nucleic acid's lower strand, creating 5' hanging ends at each end. Alternatively, the single-stranded complementary region may extend from the 3' end of the upper strand of the donor nucleic acid and from the 3' end of the lower strand of the template, creating 3' hanging ends.

[00195] Complementary regions can be of any length sufficient to promote ligation between the exogenous donor nucleic acid and the target nucleic acid. Exemplary complementary regions are from about 1 to about 5 nucleotides in length, between about 1 to about 25 nucleotides in length, or from about 5 to about 150 nucleotides in length. For example, the complementary region may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. Alternatively, the complementary region may be approximately 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110 , 110-120, 120-130, 130-140 or 140-150 nucleotides in length or more.

[00196] Such complementary regions may be complementary to hanging ends created with two pairs of nikases. Two staggered double-strand breaks can be created using a first and second nickase that cleaves opposite DNA strands to create a first double-strand break, and a third and quarter nickase that cleaves opposite DNA strands to create a second double-strand break. For example, the Cas protein can be used to create a single strand break in the first, second, third, and fourth guide RNA target sequences corresponding to the first, second, third, and fourth guide RNA. The first and second target sequences for the guide RNA can be positioned to create the first cleavage site such that nicks created by the first and second nicases on the first and second DNA strands create a double-strand break (i.e., the first cleavage site contains nicks in within the first and second target guide RNA sequences). Similarly, the third and fourth guide RNA target sequences can be positioned to create a second cleavage site such that nicks created by the third and fourth nicases on the first and second DNA strands create a double-stranded nick (i.e., the second cleavage site contains single-stranded gaps within the third and fourth target guide RNA sequences). Optionally, the nicks within the first and second guide RNA target sequences and/or the third and fourth guide RNA target sequences can be misaligned nicks that create drooping ends. The bias window may be, for example, at least about 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp. bp, 70 bp, 80 bp, 90 bp, 100 bp or more. See Ran et al. (2013) Cell 154:1380-1389; Mali et al. (2013) Nat. Biotech.31:833-838; and Shen et al. (2014) Nat. Methods 11:399-404, each of which is hereby incorporated by reference in its entirety for all purposes. In such cases, a double-stranded exogenous donor nucleic acid can be designed with single-stranded complementary regions that are complementary to the overhangs created by single-strand breaks within the first and second target guide RNA sequences and by single-strand breaks within the third and fourth target guide RNA sequences. . Such an exogenous donor nucleic acid can be inserted by non-homologous end joining mediated ligation.

(3) Donor nucleic acids for insertion by homologous repair [00197] Some exogenous donor nucleic acids contain homologous arms. If the exogenous donor nucleic acid also contains a nucleic acid insert, homologous arms may flank the nucleic acid insert. For ease of reference, homologous arms are referred to herein as 5' and 3' (ie, upstream and downstream) homologous arms. This terminology refers to the relative position of the homologous arms relative to the nucleic acid insert within the exogenous donor nucleic acid. The 5' and 3' homologous arms correspond to regions within the target genomic locus, referred to herein as "5' target sequence" and "3' target sequence", respectively.

[00198] A homologous arm and a target sequence "match" or are "matched" to each other when the two regions share a sufficient level of sequence identity with each other to act as substrates for the homologous recombination reaction. The term "homology" includes DNA sequences that are either identical or have sequence identity relative to a corresponding sequence. The sequence identity between a given target sequence and the corresponding homologous arm occurring in the exogenous donor nucleic acid may be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity between the homologous arm of the exogenous donor nucleic acid (or fragment thereof) and the target sequence (or fragment thereof) may be at least 50%, 55%, 60%, 65%, 70%, 75% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 91%, 98%, 99%, or 100% sequence identity such that the sequences undergo homologous recombination. Moreover, the corresponding region of homology between the homologous arm and the corresponding target sequence may be of any length that is sufficient to allow homologous recombination. Exemplary homologous arms are from about 25 nucleotides to about 2.5 kb. in length, from about 25 nucleotides to about 1.5 kb. in length, or from about 25 to about 500 nucleotides in length. For example, a given homologous arm (or each of the homologous arms) and/or the corresponding target sequence may contain corresponding regions of homology that are approximately 25-30, 30-40, 40-50, 50-60, 60-70, 70-80 , 80-90, 90-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450 or 450-500 nucleotides in length, so that homologous arms are characterized by sufficient homology to undergo homologous recombination with the corresponding target sequences within the target nucleic acid. Alternatively, a given homologous arm (or each homologous arm) and/or the corresponding target sequence may contain corresponding regions of homology that are from about 0.5 kbp. to about 1 kb, from about 1 kb to about 1.5 kb, from about 1.5 kb up to about 2 kb. or from about 2 kb. up to about 2.5 kb. in length. For example, each of the homologous arms may be approximately 750 nucleotides in length. Homologous arms may be symmetrical (each approximately the same size in length) or they may be asymmetrical (one longer than the other).

[00199] When the CRISPR/Cas system is used in combination with an exogenous donor nucleic acid, the 5' and 3' target sequences are not necessarily located in sufficient proximity to the Cas cleavage site (e.g., in sufficient proximity to the target sequence for the guide RNA) so as to facilitate the occurrence of a homologous recombination event between target sequences and homologous arms upon single-strand break (nick) or double-strand break at the Cas cleavage site. The term "Cas cleavage site" includes a DNA sequence in which a single-strand break or double-strand break is created by a Cas enzyme (eg, a Cas9 protein complexed with a guide RNA). Target sequences within the targeted locus that correspond to the 5' and 3' homologous arms of the exogenous donor nucleic acid are "located in sufficient proximity" to the Cas cleavage site if the distance is such as to promote a homologous recombination event between the 5' and 3' target sequences and homologous arms at a single-strand break or double-strand break at the Cas cleavage site. Thus, target sequences corresponding to the 5' and/or 3' homologous arms of the exogenous donor nucleic acid can be, for example, within at least 1 nucleotide from a given Cas cleavage site, or within at least 10 nucleotides to about 1000 nucleotides of the given cleavage site Cas. By way of example, the Cas cleavage site may be directly adjacent to at least one or both of the target sequences.

[00200] The spatial relationships of the target sequences that correspond to the homologous arms of the exogenous donor nucleic acid and the Cas cleavage site can vary. For example, the target sequences may be located 5' from the Cas cleavage site, the target sequences may be located 3' from the Cas cleavage site, or the target sequences may flank the Cas cleavage site.

B. Methods for optimizing the ability of CRISPR/Cas to cut a target genomic nucleic acid in vivo or ex vivo

[00201] Various methods are contemplated for optimizing delivery of CRISPR/Cas to a cell or non-human animal, or optimizing CRISPR/Cas activity in vivo. Such methods may include, for example, the following: (a) performing a method for testing the ability of CRISPR/Cas to modify a target genomic locus, as described above, for the first time in a first non-human animal or in a first cell; (b) changing the variable and performing the method a second time in a second non-human animal (ie the same species) or in a second cage with the changed variable; and (c) comparing the modification of the target genomic locus in step (a) with the modification of the target genomic locus in step (b), and selecting a method that results in a more efficient modification of the target genomic locus.

[00202] More efficient modification of a target genomic locus can mean different things depending on the desired effect within a non-human animal or cell. For example, a more efficient modification of a target genomic locus may mean one or more or all of the following: increased efficiency, increased accuracy, increased consistency, or increased specificity. Increased efficiency refers to increased levels of modification of a target genomic locus (eg, an increased percentage of cells targeted within a particular target cell type, within a particular target tissue, or within a particular target organ). Increased accuracy refers to a more precise modification of the target genomic locus (eg, an increased percentage of targeted cells with the same modification or with the desired modification without excessive unintentional insertions and deletions (eg, NHEJ indels)). Increased consistency refers to more consistent modification of a target genomic locus among different target cell, tissue, or organ types if more than one cell, tissue, or organ type is targeted (eg, modification of more cell types within the target organ). If a particular organ is being targeted, increased consistency can also refer to a more consistent modification at all locations within the organ. Increased specificity may refer to increased specificity for the targeted genomic locus or loci, increased specificity for the targeted cell type, increased specificity for the targeted tissue type, or increased specificity for the targeted organ. For example, increased genomic locus specificity refers to less modification of non-target genomic loci (eg, a reduced percentage of targeted cells with modifications at unintentional, non-target genomic loci instead of or in addition to modifying the target genomic locus). Similarly, increased specificity for a cell type, tissue type, or organ type refers to less modification of non-target cell types, tissue types, or organ types if a particular cell type, tissue type, or organ type is to be targeted (e.g., when a particular organ is to be targeted (e.g., liver), there is less cell modification in organs or tissues that are not intended targets).

[00203] The variable that changes may be any parameter. As one example, the altered variable may be a packaging or delivery method by which one or more or all of the guide RNA (or guide RNA packaged in AAV) and exogenous donor nucleic acid are introduced into a cell or non-human animal. Examples of delivery methods such as LNP, HDD, and AAV are disclosed elsewhere herein. For example, the altered variable may be an AAV serotype. As another example, the altered variable may be an administration route for introducing one or more or all of the guide RNA (eg, packaged in AAV) and exogenous donor nucleic acid into a cell or non-human animal. Examples of routes of administration such as intravenous, intravitreal, intraparenchymal, and nasal instillation are disclosed elsewhere herein.

[00204] As another example, the altered variable may be the concentration or amount of one or more or all of the introduced guide RNA (eg, packaged in AAV) and the introduced exogenous donor nucleic acid. As another example, the altered variable may be the concentration or amount of guide RNA introduced (eg, packaged in AAV) relative to the concentration or amount of exogenous donor nucleic acid introduced.

[00205] As another example, the variable variable may be the time of administration of one or more or all of the guide RNA (eg, packaged in AAV) and exogenous donor nucleic acid relative to the time of measurement of expression or activity of one or more reporter proteins. As another example, the altered variable may be the number of times or frequency with which one or more or all of the guide RNA (eg, packaged in AAV) and exogenous donor nucleic acid are administered. As another example, the altered variable may be the time of introduction of the guide RNA relative to the time of administration of the exogenous donor nucleic acid.

[00206] As another example, the altered variable may be a form in which one or more or all of the guide RNA and exogenous donor nucleic acid are administered. For example, the guide RNA can be administered in the form of DNA or in the form of RNA. The exogenous donor nucleic acid may be DNA, RNA, single-stranded, double-stranded, linear, circular, and so on. Likewise, each of the components may contain various combinations of modifications for stability, for reducing off-target effects, for ease of delivery, and so on. As another example, the altered variable may be one or more or all of an introduced guide RNA (eg, introducing another guide RNA with a different sequence) and an introduced exogenous donor nucleic acid (eg, introducing a different exogenous donor nucleic acid with a different sequence).

C. Introduction of guide RNAs and other components into non-human animal cells and bodies

[00207] The methods disclosed herein involve administering to a cell or non-human animal one or more or all of guide RNAs and exogenous donor nucleic acids. "Introducing" includes presenting a nucleic acid or protein to a cell or non-human animal in such a way that the nucleic acid or protein gains access to the interior of the cell or to the interior of the cells in the body of the non-human animal. Administration may be by any means, and two or more components (eg, two of the components or all of the components) may be administered to a cell or non-human animal simultaneously or sequentially in any combination. For example, the exogenous donor nucleic acid may be administered to a cell or non-human animal prior to the administration of the guide RNA, or it may be administered after the administration of the guide RNA (e.g., the exogenous donor nucleic acid may be administered about 1, 2, 3, 4, 8 , 12, 24, 36, 48, or 72 hours before or after guide RNA injection). See, for example, US 2015/0240263 and US 2015/0110762, each of which is incorporated herein by reference in its entirety for all purposes. In addition, two or more components can be administered to a cell or non-human animal by the same delivery method or by different delivery methods. Similarly, two or more components can be administered to a non-human animal by the same route of administration or by different routes of administration.

[00208] The guide RNA can be introduced into the cell in the form of RNA (eg, in vitro transcribed RNA) or in the form of DNA encoding the guide RNA. When introduced in the form of DNA, the DNA encoding the guide RNA can be operably linked to a promoter active in the cell. For example, the guide RNA can be delivered by AAV and can be expressed in vivo under the control of the U6 promoter. Such DNA may be in one or more expression constructs. For example, such expression constructs may be components of a single nucleic acid molecule. Alternatively, they may be separated in any combination among two or more nucleic acid molecules (i.e., DNA encoding one or more CRISPR RNAs and DNA encoding one or more tracrRNAs may be components of separate nucleic acid molecules).

[00209] Nucleic acids encoding guide RNAs can be operably linked to a promoter in an expression construct. Expression constructs include any nucleic acid constructs capable of directing the expression of a gene or other nucleic acid sequence of interest and that can transfer such nucleic acid sequence of interest to a target cell. Suitable promoters that can be used in an expression construct include promoters active in, for example, one or more of the following: eukaryotic cell, human cell, non-human cell, mammalian cell, non-human mammalian cell, rodent cell , mouse cell, rat cell, hamster cell, rabbit cell, pluripotent cell, embryonic stem (ES) cell, adult stem cell, developmentally restricted progenitor cell, induced pluripotent stem cell (iPS) or single cell stage embryo. Such promoters may be, for example, conditional promoters, inducible promoters, constitutive promoters or tissue-specific promoters. Optionally, the promoter may be a bidirectional promoter driving the expression of both the guide RNA in one direction and the other component in the other direction. Such bidirectional promoters may consist of (1) a complete conventional unidirectional Pol III promoter that contains 3 external controls: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second main Pol III promoter that includes PSE and a TATA box fused to the 5' end of DSE in reverse orientation. For example, in the HI promoter, DSE is adjacent to PSE and a TATA box, and the promoter can be made bidirectional by creating a hybrid promoter in which reverse transcription is controlled by adding PSE and a TATA box derived from the U6 promoter. See, for example, US 2016/0074535, incorporated herein by reference in its entirety for all purposes. The use of a bidirectional promoter for the expression of genes encoding the guide RNA and another component at the same time allows the creation of compact expression cassettes to facilitate delivery.

[00210] Exogenous donor nucleic acids and guide RNAs (or nucleic acids encoding guide RNAs) can be provided in compositions containing a carrier that enhances the stability of the exogenous donor nucleic acid or guide RNA (for example, prolonging the period under given storage conditions (for example, - 20°C, 4°C, or ambient temperature) for which degradation products remain below a threshold, such as below 0.5% by weight of the parent nucleic acid or protein, or an increase in in vivo stability). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, microspheres, D,L-lactic-glycolic acid copolymer (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules.

[00211] Provided herein are various methods and compositions for introducing a nucleic acid or protein into a non-human cell or animal. Methods for introducing nucleic acids into various cell types are known in the art and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[00212] Protocols for transfection, as well as protocols for introducing nucleic acid sequences into cells, may vary. Non-limiting methods of transfection include chemical methods of transfection; methods using liposomes; nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Set USA 1A (4): 1590-4, and Kriegler, M (1991 ) Transfer and Expression: A Laboratory Manual, New York: W. H. Freeman and Company, pp. 96-97); dendrimers or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation and optical transfection. Particle-based transfection includes the use of a gene gun or magnetic transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral techniques can also be used for transfection.

[00213] The introduction of nucleic acids or proteins into a cell can also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection. Nucleofection is an advanced electroporation technology that delivers nucleic acid substrates not only into the cytoplasm, but also across the nuclear membrane and into the nucleus. In addition, the use of nucleofection in the methods disclosed herein typically requires much fewer cells than conventional electroporation (eg, only about 2 million compared to 7 million with conventional electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[00214] The introduction of nucleic acids or proteins into a cell (eg, a zygote) can also be accomplished by microinjection. In zygotes (ie, single-cell stage embryos), microinjection can be performed into the mother's and/or father's pronucleus, or into the cytoplasm. If microinjection occurs in only one pronucleus, the paternal pronucleus is preferred due to its larger size. Alternatively, microinjection can be done by injection into both the nucleus/pronucleus and the cytoplasm: first, the needle can be inserted into the nucleus/pronucleus and the first amount can be injected, when the needle is removed from the embryo at the single cell stage, the second amount can be injected into the cytoplasm. Methods for conducting microinjection are well known. See, for example, Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); see also Meyer et al. (2010) Proc. Natl. Acad. sci. USA 107:15022-15026 and Meyer et al. (2012) Proc. Natl. Acad. sci. USA 109:9354-9359.

[00215] Other methods of introducing nucleic acid or proteins into a cell or non-human animal may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device delivery. As specific examples, the nucleic acid or protein can be administered to a non-human cell or animal in a carrier such as a poly(lactic acid) (PLA) microsphere, a D,L-lactic-glycolic acid copolymer (PLGA) microsphere, liposome, micelle, reverse micelle, lipid cochleate, or lipid microtubule. Some specific examples of delivery to a non-human animal include hydrodynamic delivery, virus-mediated delivery (eg, adeno-associated virus (AAV) mediated delivery), and lipid nanoparticle-mediated delivery.

[00216] The introduction of nucleic acids and proteins into the cells or body of non-human animals can be accomplished using hydrodynamic delivery (HDD). Hydrodynamic delivery has emerged as a method for intracellular delivery of DNA in vivo. To deliver genes to parenchymal cells through a chosen blood vessel, only the required DNA sequences need to be injected, eliminating the safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is able to reach cells in various tissues accessible to the blood. Hydrodynamic delivery uses the force generated by rapidly injecting a large volume of solution into incompressible blood in the circulation to overcome the physical barriers of the endothelium and cell membranes that prevent large and membrane-impermeable compounds from entering parenchymal cells. In addition to DNA delivery, this method is applicable for efficient intracellular delivery of RNA, proteins and other small compounds in vivo. See, for example, Bonamassa et al. (2011) Pharm. Res. 28(4):694-701, incorporated herein by reference in its entirety for all purposes.

[00217] Administration of nucleic acids can also be accomplished by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses/viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. Viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. Viruses may integrate into the host genome or, alternatively, may not integrate into the host genome. Such viruses can also be developed to reduce immunity. Viruses may be capable of replication or may be replication defective (eg, defective in one or more genes required for additional rounds of replication and/or virion packaging). Viruses can cause transient expression, long-term expression (eg, for at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or persistent expression (eg, Cas9 and/or gRNA). Exemplary virus titers (eg, AAV titers) include 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , and 10 ¹⁶ vector genomes/ml.

[00218] The AAV ssDNA genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow the synthesis of a complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between two ITRs and Rep and Cap can be delivered in transpositions. In addition to Rep and Cap, AAV may require a helper plasmid containing adenovirus genes. These genes (E4, E2a and VA) mediate AAV replication. For example, a transfer plasmid, Rep/Cap, and a helper plasmid can be transfected into HEK293 cells containing the adenovirus E1+ gene to generate infectious AAV particles. Alternatively, Rep, Cap, and adenovirus helper genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses such as retroviruses.

[00219] Numerous AAV serotypes have been identified. These serotypes differ in the cell types they infect (ie, their tropism), which allows for preferential transduction of certain cell types. CNS tissue serotypes include AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9. Serotypes for heart tissue include AAV1, AAV8 and AAV9. Renal tissue serotypes include AAV2. Lung tissue serotypes include AAV4, AAV5, AAV6 and AAV9. Pancreatic tissue serotypes include AAV8. Serotypes for photoreceptor cells include AAV2, AAV5 and AAV8. Retinal pigment epithelial tissue serotypes include AAV1, AAV2, AAV4, AAV5 and AAV8. Skeletal muscle tissue serotypes include AAV1, AAV6, AAV7, AAV8 and AAV9. Liver tissue serotypes include AAV7, AAV8 and AAV9 and in particular AAV8.

[00220] Tropism can be further refined using pseudotyping, which is the mixing of capsid and genome from different viral serotypes. For example, AAV2/5 indicates a virus containing a serotype 2 genome packaged in a capsid from serotype 5. The use of pseudotyped viruses can increase transduction efficiency as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to modify viral tropism. For example, AAV-DJ contains a hybrid capsid of eight serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that exhibits the properties of AAV-DJ, but with improved brain uptake. AAV serotypes can also be modified through mutations. Examples of AAV2 mutations include Y444F, Y500F, Y730F and S662V. Examples of AAV3 mutations include Y705F, Y731F and T492V. Examples of AAV6 mutations include S663V and T492V. Other pseudotyped/modified AAV variants include AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5, AAV8.2, and AAV/SASTG.

[00221] Self-complementary AAV variants (scAAV) can be used to accelerate transgene expression. Because AAV depends on the cell's DNA replication machinery to synthesize the complementary strand of the AAV single-stranded DNA genome, transgene expression can be delayed. To eliminate this delay, scAAVs containing complementary sequences that are capable of spontaneous annealing upon infection can be used, eliminating the need for host cell DNA synthesis. However, single stranded AAV vectors (ssAAV) can also be used.

[00222] To increase packaging capacity, longer transgenes can be split between two AAV transfer plasmids, the first with a 3' splice donor site and the second with a 5' splice acceptor site. When cells are co-infected, these viruses form concatemers, splice together, and a full-length transgene can be expressed. While this allows for increased expression of the transgene, expression is less efficient. Homologous recombination is used in such capacity increasing methods. For example, a transgene can be split between two transfer plasmids, but with significant sequence overlap such that co-expression induces homologous recombination and full-length transgene expression.

[00223] Administration of nucleic acids and proteins can also be accomplished by lipid nanoparticle (LNP) mediated delivery. For example, LNP-mediated delivery can be used to deliver guide RNA in the form of RNA. Delivery via such methods results in the transient presence of the guide RNA and the biodegradable lipids improve clearance, improve tolerability and reduce immunogenicity. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles containing many lipid molecules physically connected to each other by intermolecular forces. These include microspheres (including single-layer and multilayer vesicles such as liposomes), dispersed phase in emulsion, micelles, or internal phase in suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Compositions that contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, accessory lipids that enhance transfection, and stealth lipids that increase the length of time the nanoparticles can exist in vivo. . Examples of suitable cationic lipids, neutral lipids, anionic lipids, accessory lipids and invisible lipids can be found in International Patent Publication WO 2016/010840 A1, incorporated herein by reference in its entirety for all purposes. An exemplary lipid nanoparticle may contain a cationic lipid and one or more other components. In one example, the other component may contain an auxiliary lipid such as cholesterol. In another example, other components may contain an auxiliary lipid such as cholesterol and a neutral lipid such as DSPC. In another example, other components may contain an accessory lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033.

[00224] The LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal release; (ii) a neutral lipid for stabilization; (iii) an auxiliary lipid for stabilization; and (iv) invisible lipid. See, for example, Finn et al. (2018) Cell Reports 22:1-9 and WO 2017/173054 Al, each of which is incorporated herein by reference in its entirety for all purposes. In some LNPs, the cargo may include a guide RNA or a nucleic acid encoding a guide RNA. In some LNPs, the cargo may include a guide RNA or a nucleic acid encoding a guide RNA and an exogenous donor nucleic acid.

[00225] The lipid for encapsulation and endosomal release may be a cationic lipid. The lipid may also be a biodegradable lipid, such as a biodegradable ionizable lipid. One example of a suitable lipid is lipid A or LP01 which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl )oxy)methyl)propyl-octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl )propyl(9Z,12Z)-octadeca-9,12-dienoate. See, for example, Finn et al. (2018) Cell Reports 22:1-9 and International Patent Publication WO 2017/173054 A1, each document herein incorporated by reference in its entirety for all purposes. Another example of a suitable lipid is lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), which is also is called ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Another example of a suitable lipid is lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diyl(9Z,9'Z,12Z ,12'Z)-bis(octadeca-9,12-dienoate). Another example of a suitable lipid is lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl-3-octylundecanoate. Other suitable lipids include heptatriaconta-6,9 ,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (also known as Dlin-MC3-DMA (MC3))).

[00226] Some such lipids suitable for use in the LNPs described herein are biodegradable in vivo. For example, LNPs containing such a lipid include those in which at least 75% of the lipid is cleared from plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. As another example, at least 50% of the LNP is cleared from plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days.

[00227] Such lipids may be ionizable depending on the pH of the environment in which they are found. For example, in a slightly acidic environment, lipids can be protonated and thus carry a positive charge. Conversely, in a weakly basic environment such as, for example, blood, where the pH is approximately 7.35, lipids may not be protonated and therefore carry a charge. In some embodiments, lipids can be protonated at a pH of at least about 9, 9.5, or 10. The ability of such a lipid to carry a charge is related to its own pKa. For example, a lipid may independently have a pKa value ranging from about 5.8 to about 6.2.

[00228] Neutral lipids function to stabilize and improve LNP processing. Examples of suitable neutral lipids include a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (PEC), dilauriloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine ( MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine ( SPPC), 1,2-diacosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine amine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. For example, the neutral phospholipid can be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

[00229] Accessory lipids include lipids that enhance transfection. The mechanism by which the accessory lipid enhances transfection may involve increasing the stability of the particles. In some cases, an auxiliary lipid can enhance membrane fusogenicity. Accessory lipids include steroids, sterols, and alkylresorcinols. Examples of suitable auxiliary lipids suitable for use include cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate. In one example, the accessory lipid may be cholesterol or cholesterol hemisuccinate.

[00230] Stealth lipids include lipids that alter the length of time that nanoparticles can exist in vivo. Stealth lipids can assist the formulation process, for example, by reducing particle aggregation and controlling particle size. Invisible lipids can modulate the pharmacokinetic properties of LNP. Suitable stealth lipids include lipids with a hydrophilic head associated with a lipid moiety.

[00231] The hydrophilic head of the stealth lipid may comprise, for example, a polymer moiety selected from PEG-based polymers (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly( N-vinylpyrrolidone), polyamino acids and poly(N-(2-hydroxypropyl)methacrylamide). The term PEG means any polyethylene glycol or other polyalkylene ether polymer. In some formulations, the LNP PEG is PEG-2K, also referred to as PEG 2000, which has an average molecular weight of approximately 2000 daltons. See, for example, International Patent Publication WO 2017/173054 A1, incorporated herein by reference in its entirety for all purposes.

[00232] A stealth lipid moiety can be derived from, for example, diacylglycerol or diacylglycamide, including compounds containing a dialkylglycerol or dialkylglycamide group with an alkyl chain length independently containing from about C4 to about C40 saturated or unsaturated carbon atoms, where the chain may contain one or more functional groups such as, for example, an amide or an ester. The dialkylglycerol or dialkylglycamide group may additionally contain one or more substituted alkyl groups.

[00233] As one example, a stealth lipid can be selected from the following: PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycerol, PEG -dipalmitoylglycamide and PEG-distearoylglycamide, PEG-cholesterol ethylene glycol), PEG-DMB (3,4-ditradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k- DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA) and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA).In one specific example, the invisible lipid may be PEG2k-DMG.

[00234] LNPs may contain various appropriate molar ratios of lipid components in the formulation. The mol% ratio of CCD lipid may be, for example, from about 30 mol% to about 60 mol%, from about 35 mol% to about 55 mol%, from about 40 mol% to about 50 mol%, from about 42 mol% to about 47 mol% or about 45%. The mol% ratio of accessory lipid may be, for example, from about 30 mol% to about 60 mol%, from about 35 mol% to about 55 mol%, from about 40 mol% to about 50 mol%, from about 41 mol% to about 46 mol%, or about 44 mol%. The mol% neutral lipid ratio may be, for example, from about 1 mol% to about 20 mol%, from about 5 mol% to about 15 mol%, from about 7 mol% to about 12 mol%, or about 9 mol%. The mole% ratio of the stealth lipid can be, for example, about 1 mole% to about 10 mole%, about 1 mole% to about 5 mole%, about 1 mole% to about 3 mole%, about 2 mole%, or about 1 mol%.

[00235] LNPs can have different ratios between the positively charged amine groups of the biodegradable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the N/P ratio. For example, the N/P ratio may be from about 0.5 to about 100, from about 1 to about 50, from about 1 to about 25, from about 1 to about 10, from about 1 to about 7, from about 3 to about 5, about 4 to about 5, about 4, about 4.5, or about 5.

[00236] In some LNPs, the cargo may contain exogenous donor nucleic acid and gRNA. Exogenous donor nucleic acid and gRNA can be in various ratios. For example, the LNP composition may include a ratio of exogenous donor nucleic acid to gRNA nucleic acid ranging from about 25:1 to about 1:25, ranging from about 10:1 to about 1:10, ranging from about 5:1 to about 1:5, or about 1:1. Alternatively, the composition of the LNP may include a ratio of exogenous donor nucleic acid to gRNA nucleic acid from about 1:1 to about 1:5, about 5:1 to about 1:1, about 10:1, or about 1:10. Alternatively, the composition of the LNP may include a ratio of exogenous donor nucleic acid to gRNA nucleic acid of approximately 1:10, 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1: 5, 1:10 or 1:25.

[00237] A specific example of a suitable LNP has a nitrogen to phosphate (N/P) ratio of 4.5 and contains biodegradable cationic lipid, cholesterol, DSPC and PEG2k-DMG in a molar ratio of 45:44:9:2. The biodegradable cationic lipid may be (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propylooctadeca- 9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-(9Z,12Z )-octadeca-9,12-dienoate. See, for example, Finn et al. (2018) Cell Reports 22:1-9, incorporated herein by reference in its entirety for all purposes. Another specific example of a suitable LNP contains Dlin-MC3-DMA (MC3), cholesterol, DSPC and PEG-DMG in a molar ratio of 50:38.5:10:1.5.

[00238] The delivery method can be chosen to reduce immunogenicity. For example, gRNA and exogenous donor nucleic acid can be delivered in different ways (eg, bimodal delivery). These different methods can confer different pharmacodynamic or pharmacokinetic properties to the target molecule of interest (eg, gRNA or nucleic acid encoding or exogenous donor nucleic acid/repair template). For example, different methods may result in different tissue distributions, different half-lives, or different time distributions. Some delivery methods (eg, delivery of a nucleic acid vector that is maintained in the cell through autonomous replication or genomic integration) result in more permanent expression and presence of the molecule, while other delivery methods are transient and less permanent (eg, RNA or protein delivery). Delivery of the components in a more transient manner, eg as RNA or protein, can ensure that the Cas/gRNA complex is present and active only for a short period of time, and can reduce immunogenicity. Such temporary delivery may also reduce the likelihood of off-target modifications.

[00239] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Methods of systemic administration include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous infusion. Methods of topical administration include, for example, intrathecal, intracerebroventricular, intraparenchymal (eg, localized intraparenchymal delivery to the following: striatum (eg, caudate nucleus or putamen), cerebral cortex, precentral gyrus, hippocampus (eg, dentate gyrus or area SAZ), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla oblongata, hypothalamus, midbrain roof, midbrain tegmentum, or substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral pathways. Significantly lower amounts of components (compared to systemic approaches) may be effective when administered topically (eg, intraparenchymal or intravitreal) compared to systemic administration (eg, intravenously). Topical routes of administration may also reduce or eliminate the occurrence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

[00240] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. A specific example is intravenous infusion. Nasal instillation and intravitreal injection are other specific examples. Compositions containing guide RNAs (or nucleic acids encoding guide RNAs) can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or additives. The composition may depend on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or additive is compatible with the other ingredients of the formulation and is not substantially harmful to its recipient.

[00241] The frequency of administration and number of dosages may depend on the half-life of the exogenous donor nucleic acids or guide RNAs (or nucleic acids encoding guide RNAs) and the route of administration, among other factors. The introduction of nucleic acids or proteins into a cell or non-human animal can be performed once or several times over a period of time. For example, the administration can be performed at least twice during a period of time, at least three times during a period of time, at least four times during a period of time, at least five times during a period of time, at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of time, at least ten times over a period of time, at least eleven times, at least twelve times in a period of time, at least thirteen times in a period of time, at least fourteen times in a period of time, at least fifteen times in a period of time, at least sixteen times in a period of time time, at least seventeen times during a period of time, at least eighteen times during a period of time, at least nineteen times during a period of time e period of time or at least twenty times within a period of time.

D. Measurement of CRISPR/Cas activity in vivo and evaluation of target genomic locus modification

[00242] The methods disclosed herein may further include assessing the modification of a target genomic locus. Methods for detecting or measuring expression or activity will depend on the target genomic locus being modified.

[00243] For example, if a target genomic locus contains a gene encoding an RNA or protein, and the intended modification is to change the expression of the encoded RNA or protein, a method for evaluating the modification of the target genomic locus may include measuring the expression or activity of the encoded RNA or protein. For example, if the encoded protein is a serum-released protein, the serum levels of the encoded protein can be measured. Assays for measuring RNA and protein levels and activities are well known.

[00244] Alternatively, the methods disclosed herein may further comprise identifying a cell containing a modified target genomic locus in which the sequence has been modified with non-homologous end joining (e.g., the presence of small inserts or deletions (indels)) after CRISPR/ Cas in which the sequence at the target genomic locus between two target guide RNA sequences has been excised or in which the target genomic locus has been modified by recombination with an exogenous donor nucleic acid. Various methods can be used to identify cells with targeted genetic modification. Screening may include a quantitative analysis to assess the modification of the allele (MOA) of the parent chromosome. For example, quantitative analysis can be performed using quantitative PCR, such as real-time PCR (qPCR). Real-time PNR may use a first primer set that recognizes a target locus and a second primer set that recognizes a non-target reference locus. The primer set may contain a fluorescent probe that recognizes the amplified sequence. Other examples of suitable assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probe(s), INVADER® probes, TAQMAN® Molecular Beacon probes, or probe technology. ECLIPSE™ (see, for example, US 2005/0144655, incorporated herein by reference in its entirety for all purposes).

[00245] Next generation sequencing (NGS) can also be used for screening. Next generation sequencing may also be referred to as "NGS" or "massive parallel sequencing" or "high throughput sequencing". NGS can be used as a screening tool in addition to MOA assays to determine the exact nature of the targeted genetic modification and whether it is consistent across cell types or tissue types or organ types.

[00246] Modification of a target genomic locus in a non-human animal can be assessed in any cell type from any tissue or organ. For example, modification of a target genomic locus can be assessed in multiple cell types from the same tissue or organ, or in cells from multiple locations in a tissue or organ. This can provide information about which cell types in the target tissue or organ are being modified, or which regions of the tissue or organ are being reached by CRISPR/Cas and being modified. As another example, modification of a target genomic locus can be assessed in multiple tissue types or multiple organs. In methods that target a particular tissue or organ, this can provide information on how effective the target is on that tissue or organ and whether there are off-target effects in other tissues or organs.

[00247] In some specific examples of non-human animals expressing Cas9, it can be used to estimate the editing frequency of various guide RNAs. Guide RNAs can be administered either as a single guide RNA (modified and unmodified) or duplex RNA or expressed under the control of the U6 promoter (eg via AAV). Cas9 expressing non-human animals can also be bred with non-human animals, including non-human animals with humanized alleles expressing guide RNAs, for evaluation in disease models.

IV. Methods for producing non-human animals containing a Cos expression cassette and/or a recombinase expression cassette

[00248] Various methods are contemplated for producing a non-human animal comprising one or more or all of a Cas expression cassette and a recombinase expression cassette, as disclosed elsewhere herein. Any conventional method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, for example, Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is incorporated herein by reference in its entirety for all purposes. Such genetically modified non-human animals can be generated, for example, by knocking a gene at a target locus (eg, a safe harbor locus such as Rosa26) or by using a randomly integrated transgene. See, for example, International Patent Publications WO 2014/093622 and WO 2013/176772, each of which is incorporated herein by reference in its entirety for all purposes. Methods for targeting a construct to the Rosa26 locus are described, for example, in US 2012/0017290, US 2011/0265198 and US 2013/0236946, each of which is incorporated herein by reference in its entirety for all purposes.

[00249] For example, a method for producing a non-human animal comprising one or more or all of a Cas expression cassette and a recombinase expression cassette as disclosed elsewhere herein may include: (1) modifying the genome of a pluripotent cell to include one or some or all of the Cas expression cassette and the recombinase expression cassette; (2) identifying or selecting a genetically modified pluripotent cell containing one or more or all of the Cas expression cassette and the recombinase expression cassette; (3) introducing the genetically modified pluripotent cell into a non-human host animal embryo; and (4) implantation and gestation of the host embryo in the body of a surrogate mother. Optionally, a host embryo containing a modified pluripotent cell (eg, a non-human ES cell) may be incubated to the blastocyst stage prior to implantation and gestation in a surrogate mother to produce a non-human F0 animal. The surrogate mother can then produce a non-human F0 generation animal containing one or more or all of the Cas expression cassette and the recombinase expression cassette.

[00250] The methods may further comprise identifying a cell or animal with a modified target genomic locus. Various methods can be used to identify cells and animals with targeted genetic modification.

[00251] The screening step may include, for example, a quantitative analysis to assess the allele modification (MOA) of the parent chromosome. For example, quantitative analysis can be performed using quantitative PCR, such as real-time PCR (qPCR). Real-time PCR can use a first primer set that recognizes a target locus and a second primer set that recognizes a non-target reference locus. The primer set may contain a fluorescent probe that recognizes the amplified sequence.

[00252] Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probe(s), INVADER® probes, TAQMAN® Molecular Beacon probes or ECLIPSE™ probe technology (see, for example, US 2005/0144655, incorporated herein by reference in its entirety for all purposes).

[00253] An example of a suitable pluripotent cell is an embryonic stem (ES) cell (eg, mouse ES cell or rat ES cell). A modified pluripotent cell can be generated, for example, by the following: (a) introducing into the cell one or more targeting vectors containing an insert nucleic acid flanked by 5' and 3' homologous arms corresponding to the 5' and 3' target sites, wherein the nucleic acid the insert contains one or more or all of the Cas expression cassette and the recombinase expression cassette; and (b) identifying at least one cell containing in its genome an insert nucleic acid integrated at a target genomic locus. Alternatively, a modified pluripotent cell can be prepared as follows: (a) introducing into the cell the following: (i) a nuclease agent, wherein the nuclease agent induces a single-strand break or a double-strand break on a target sequence within the target genomic locus; and (ii) one or more targeting vectors comprising an insert nucleic acid flanked by 5' and 3' homologous arms corresponding to the 5' and 3' target sites located in sufficient proximity to the target sequence, the insert nucleic acid comprising one or some or all of the Cas expression cassette and the recombinase expression cassette; and (c) identifying at least one cell containing the modification (eg, integration of the insert nucleic acid) at the target genomic locus. You can use any nuclease agent that causes a single-strand break or double-strand break in the desired target sequence. Examples of suitable nucleases include a transcription activator-like effector nuclease (TALEN), a zinc finger domain nuclease (ZFN), a meganuclease, and clustered regularly interspersed short palindromic repeat (CRISPRyCRISPR-associated proteins (Cas)) systems or components of such a system (e.g., CRISPR/Cas9) See, for example, US 2013/0309670 and US 2015/0159175, each of which is hereby incorporated by reference in its entirety for all purposes.

[00254] The donor cell can be introduced into the host embryo at any stage, such as the blastocyst stage or premorula stage (ie, 4 cell stage or 8 cell stage). Offspring are created that can pass on the genetic modification through the germline. See, for example, US Pat. No. 7,294,754, incorporated herein by reference in its entirety for all purposes.

[00255] Alternatively, a method for producing non-human animals described elsewhere herein may include the following: (1) modifying the genome of a single cell stage embryo to include one or more or all of the Cas expression cassette and the recombinase expression cassette with using the methods described above to modify pluripotent cells; (2) selection of genetically modified embryos; and (3) implantation and gestation of a genetically modified embryo in the body of a surrogate mother. Offspring are created that can pass on the genetic modification through the germ line.

[00256] Nuclear transfer techniques can also be used to produce non-human mammals. Briefly, nuclear transfer methods may include the following steps: (1) enucleating an oocyte or obtaining an enucleated oocyte; (2) isolating or obtaining a donor cell or nucleus to combine with the enucleated oocyte; (3) inserting a cell or nucleus into an enucleated oocyte to form a remodeled cell; (4) implanting the reconstructed cell into the uterus of the animal to form an embryo; and (5) ensuring the development of the embryo. In such methods, oocytes are typically recovered from deceased animals, although they may also be isolated from the oviducts and/or ovaries of living animals. Oocytes can mature in a variety of well known media prior to enucleation. Enucleation of the oocyte can be accomplished by a number of well known methods. Insertion of a donor cell or nucleus into an enucleated oocyte to form a remodeled cell can be accomplished by microinjection of the donor cell under the vitellin layer prior to fusion. Fusion can be induced by applying a direct current electrical pulse across the contact/fusion plane (electrofusion), by exposing the cells to fusion-promoting chemicals such as polyethylene glycol, or by an inactivated virus such as Sendai virus. The reconstructed cell may be activated by electrical and/or non-electrical means before, during and/or after the fusion of the donor nucleus and the recipient oocyte. Activation methods include electrical impulses, chemically induced shock, penetration by spermatozoa, increase in the content of divalent cations in the oocyte, and decrease in phosphorylation of cellular proteins (as with kinase inhibitors) in the oocyte. Activated remodeled cells or embryos can be cultured in well known media and then transferred into the uterus of an animal. See, for example, US 2008/0092249, WO 1999/005266, US 2004/0177390, WO 2008/017234, and US Pat. No. 7,612,250, each of which is incorporated herein by reference in its entirety for all purposes.

[00257] The various methods provided herein allow the generation of a non-human genetically modified F0 animal, wherein the cells of the genetically modified F0 animal contain one or more or all of a Cas expression cassette and a recombinase expression cassette. It is recognized that depending on the method used to produce the F0 animal, the number of cells in the F0 animal that contain one or more or all of the Cas expression cassette and the recombinase expression cassette will vary. Introduction of donor ES cells into a premorula embryo from an appropriate organism (e.g., an 8-cell stage mouse embryo), for example, using the VELOCLMOUSE® method, allows a greater percentage of the cell population of an F0 animal to be obtained that contains cells containing a nucleotide sequence of interest containing targeted genetic modification. For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cellular contribution of the non-human F0 animal may contain a cell population containing the target modification

[00258] The cells of the genetically modified F0 animal may be heterozygous for one or more or all of the Cas expression cassette and the recombinase expression cassette, or may be homozygous for one or more or all of the Cas expression cassette and the recombinase expression cassette.

[00259] All patent applications, websites, other publications, serial numbers, and the like, above or below, are incorporated by reference in their entirety for all purposes to the same extent as if it were specifically and individually indicated that each individual element incorporated herein by reference. If different versions of the sequence are associated with the registration number at different times, the version associated with the registration number on the filing date of this application is assumed. Effective filing date means the earlier of the actual filing date or the filing date of the priority application, with reference to the registration number, if applicable. Similarly, if different versions of a publication, website, or the like are published at different times, the latest version published on the actual date of submission is assumed, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention may be used in conjunction with any other, unless specifically noted otherwise. Although the present invention has been described in more detail by way of illustration and example for clarity and understanding, it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

Brief description of the sequences

[00260] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and a three-letter code for amino acids. Nucleotide sequences follow standard convention, starting at the 5' end of the sequence and moving forward (ie, left to right on each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but a complementary strand is considered to be included in any reference to the displayed strand. When a nucleotide sequence is presented encoding an amino acid sequence, it is understood that codon-degenerate variants thereof are also presented which encode the same amino acid sequence. Amino acid sequences follow standard convention, starting at the amino end of the sequence and moving forward (ie, left to right on each line) to the carboxy end.

[00261] Table 3. Description of sequences.

EXAMPLES

Example 1 Validation of Cas9 Expressing Mice

[00262] CRISPR/Cas9, an RNA-guided DNA endonuclease, catalyzes the creation of a DNA double-strand break (DSB) at the binding site of its guide RNA. An exemplary guide RNA may consist of a 42 nucleotide CRISPR RNA (crRNA) that couples to an 87 nucleotide transactivating RNA (tracrRNA). tracrRNA is complementary and base paired with srRNA, forming a functional crRNA/tracrRNA guide. This duplex RNA becomes associated with the Cas9 protein to form an active ribonucleoprotein (RNP), which can examine the genome in detail for complementarity with the 20-bp guide portion of the srRNA. A secondary requirement for strand breaking is that the Cas9 protein must recognize a protospacer adjacent motif (PAM) immediately adjacent to a sequence complementary to the cRNA guide portion (srRNA target sequence). Alternatively, an active RNP complex can also be formed by replacing the crRNA/tracrRNA duplex with a single guide RNA (sgRNA) formed by covalently linking the crRNA and tracrRNA. Such an sgRNA can be formed, for example, by fusing the 20 bp srRNA guide portion directly to a processed tracrRNA sequence. sgRNA can interact with both the Cas9 protein and DNA in the same way and with the same efficiency as the crRNA/tracrRNA duplex. The bacterial natural defense mechanism CRISPR has been shown to function effectively in mammalian cells and activate rupture-induced endogenous repair pathways. When a double strand break occurs in the genome, repair pathways will attempt to repair the DNA either by canonical or alternative non-homologous end joining (NHEJ) pathways or by homologous recombination, also called homologous repair (HDR), if an appropriate template is available. The present inventors can use these pathways to facilitate site-specific deletion of genomic regions or insertion of exogenous DNA or HDR into mammalian cells.

[00263] The CRISPR/Cas9 system is a powerful tool for genome engineering. However, one of the limitations of the system for use in vivo is the need for the simultaneous introduction of all components into a living organism. A typical way to introduce these components into cells is to transiently transfect DNA constructs into cells that will generate the appropriate RNA and protein. Although this approach is effective, it has an inherent disadvantage because cells must rely on plasmid DNA constructs to first undergo transcription and then translation before the Cas9 protein becomes available to interact with the sgRNA component. The present inventors believe that the Cas9 induced mutation rate and recombination rate can be significantly improved in the presence of a constitutively available protein.

[00264] The wild-type Cas9 coding sequence (CDS) was codon-optimized for expression in mice. An N-terminal single nuclear localization signal (NLS), a C-terminal double NLS, and a C-terminal P2A-coupled GFP fluorescent reporter were then included. the Cas9 expression cassette (MAID2599) is depicted in Figures 1 and 14 and SEQ ID NO: 1. P2A-GFP can be used to better track Cas9 expression in vivo. These components were engineered into the first intron of the Rosa26 locus of the mouse genome, together with a preceding loxP flanked neomycin resistance cassette (neo cassette) with appropriate splicing signals and a strong polyadenylation signal (polyA). The components of the Cas9 expression cassette from 5' to 3' are shown in Table 4, and the components of the Cas9 allele after removal of the 1xP flanked neomycin cassette are shown in Table 5. The Cas9-P2A-eGFP protein sequence encoded by the allele is shown in SEQ ID NO: 13.

[00265] Table 4. Components of the MAID2599 Cas9 allele.

[00266] Table 5. Components of the MAID2600 Cas9 allele.

[00267] Prior to removal of the cassette by Cre recombinase, the neomycin resistance gene will typically be efficiently transcribed and translated; however, CDS Cas9 is not normally expressed due to the presence of a strong poly(A) region that can effectively block end-to-end transcription. After removal of the neomycin cassette by Cre recombinase, the fusion mRNA for the Cas9 and GFP proteins is typically expressed constitutively via the Rosa26 promoter. Targeted cells before and after removal of the neomycin cassette were first verified by screening for allele loss to detect a single site-specific integration of the targeting vector at the Rosa26 locus. Cas9 and GFP expression was validated by isolating total RNA from targeted mESCs followed by reverse transcription to generate cDNA and TAQMAN® qPCR to detect reverse transcribed cDNA (RT-qPCR). Overall, the designed system is able to continuously or conditionally express constant levels of the Cas9 protein (requiring removal of the neomycin resistance cassette) in mESCs and mice derived from them.

[00268] Another version was developed without P2A-eGFP (MAID2660). See Figure 14. The components of the Cas9 expression cassette 5' to 3' are shown in Table 6, and the components of the Cas9 allele after removal of the loxP-flanked neomycin cassette are shown in Table 7. The Cas9 protein sequence encoded by the allele is shown in SEQ ID NO: 19.

[00269] Table 6. Components of the MAID2660 Cas9 allele.

[00270] Таблица 7. Компоненты аллеля MAID2661 Cas9.

[00270] Table 7. Components of the MAID2661 Cas9 allele.

[00271] In addition, two versions were developed with exogenous CAGG promoters and 3xFLAG tag sequences. The first version included P2A-eGFP (MAID2658) and the second version was developed without P2A-eGFP (MAID2672). See Figure 14. The components of the first version of the CAGG-Cas9 expression cassette from 5' to 3' are shown in Table 8, and the components of the first version of the CAGG-Cas9 allele after removal of the loxP-flanked neomycin cassette are shown in Table 9. Protein sequence of 3xFLAG-Cas9 The -P2A-eGFP encoded by this allele is shown in SEQ ID NO: 16. The components of the second version of the CAGG-Cas9 expression cassette 5' to 3' are shown in Table 10, and the components of the second version of the CAGG-Cas9 allele after deletion of the loxP-flanked neomycin cassettes are shown in Table 11. The 3xFLAG-Cas9 protein sequence encoded by this allele is shown in SEQ ID NO: 22.

[00272] Table 8. Components of the MAID2658 Cas9 allele.

[00273] Table 9. Components of the MAID2659 Cas9 allele.

[00274] Table 10. Components of the MAID2672 Cas9 allele.

[00275] Table 11. MAID2673 Cas9 allele components.

[00276] To validate the MAID2599/MAID2600 mESC Cas9 system with and without neomycin cassette (MAID2599 and MAID2600, respectively), two sgRNAs were transfected targeting the start and stop codon regions of the first target gene. See figure 2A. The efficiency of Cas9 cleavage was then assessed by an allele loss screen to assess the proportion of mESC clones with insertion-deletion mutations in Cas9 cleavage sites targeted by gRNA. The proportion of mESC clones in which DNA between Cas9 cleavage sites was deleted at one or both of the target alleles was also determined, causing a null mutation. Cas9 was only able to induce these genomic changes when the neomycin cassette and poly(A) (stop sequence) (MAID2600) were removed. To better evaluate genome editing capabilities for homologous repair, sgRNA targeting a second target gene was introduced along with a single stranded oligodeoxynucleotide (ssODN) as a point mutation donor. See Figure 2B. The constitutive Cas9 expression system described herein was compared to conventional methods for introducing Cas9 and sgRNA via plasmids along with ssODN. The Cas9 expression system described herein, in combination with an sgRNA expression plasmid, was able to activate nip-induced endogenous repair pathways to turn on the desired point mutation at a frequency that was equal to when both Cas9 and sgRNA were expressed from exogenous plasmids. However, when the Cas9 expression system described herein was combined with directly delivered sgRNA, it induced homology-directed microinjection mutagenesis almost twice as effective as plasmid delivery methods.

[00277] To determine the potency of endogenously expressed Cas9 in live mice, these targeted mESCs were microinjected into 8-cell mouse embryos using the VELOCIMOUSE® method. See, for example, US Pat. Nos. US 7,576,259; US 7659442; US 7294754; US Patent Application No. 2008/007800; and Poueymirou et al. (2007) Nature Biotech. 25(l):91-99, each of which is incorporated herein by reference in its entirety for all purposes. In particular, a small hole was created in the vitelline layer to facilitate the injection of targeted mESC. These injected 8 cell embryos were transferred to surrogate mothers to produce live mice carrying the transgene. After pregnancy in a surrogate mother, the injected embryos produced F0 mice that carry no detectable host embryonic contribution. Mice completely derived from ES cells were normal, healthy and fertile (germline transmission). Tissue was harvested from cassette-deleted F0 mice (MAID2600) for GFP imaging, Cas9 mRNA expression, and Cas9 protein expression. See Figures 3A-3F (bright-field and GFP imaging) and Figures 4A-4C (Cas9 mRNA expression in Figures 4A and 4C and protein expression in Figure 4B). Figure 3D shows eGFP expression in Rosa26Cas9 heterozygous mice (MAID2600), but there is no corresponding eGFP expression in wild-type mice in the liver. Figure 3F shows eGFP expression in mice heterozygous for Rosa26Cas9, but there is no corresponding eGFP expression in wild-type mice in the brain. Similarly, Cas9 mRNA expression assayed by RT-qPCR was observed in Rosa26Cas9 heterozygous mice in the brain, heart, kidney, liver, lung, quadriceps, spleen, and thymus, but Cas9 mRNA expression was not observed in corresponding tissues from wild animals. , like mice. See figures 4A and 4C. In experiments, equal mass amounts of RNA from each tissue were analyzed by RT-qPCR. The data show that Cas9 expressing mice express Cas9 mRNA at an easily detectable level in all tissues. Various tissues were collected from Cas9 expressing mice. Three tissues were collected from fourteen mice and five more tissues were collected from four mice to assess for mouse-to-mouse as well as tissue-to-tissue differences within the mouse. RNA was extracted from each of these tissues. Genomic DNA was subjected to decomposition in such a way that it was not taken into account in the qPCR reaction. The RNA was subjected to reverse transcription and then a Cas9 specific assay was used to detect Cas9 transcripts. As expected, Cas9 mice showed significant expression (ct values below 30), while WT mice showed ct values of 30 and above, indicating a lack of endogenous Cas9 protein expression.

[00278] Similarly, Cas9 protein expression determined by Western blotting using ThermFisher Cas9 MA5-23519 antibody at a 1:250 dilution and using actin as control showed Cas9 protein expression in Rosa26Cas9 heterozygous mice (MAID2600) in the spleen , liver, and brain, while the Cas9 protein was not observed in the same tissues in wild-type mice. See figure 4 B. All three tests showed a constant level of expression in all studied tissues.

[00279] An experiment was then performed to knock out target gene 3, which encodes a protein secreted by the liver and found in serum, by introducing sgRNA into primary hepatocytes isolated from mice with a removed Cas9 cassette (MAID2600) via lipid nanoparticle (LNP) delivery. See Figure 5B. As a control, the same sgRNA administration methods were paired with exogenous Cas9 expression in primary hepatocytes isolated from wild-type (WT) mice. See figure 5A. Non-homologous end joining was then assessed by next generation sequencing (NGS) to measure indel frequency at target gene locus 3. There were three conditions in the experiment: (1) LNP mediated delivery of GFP mRNA and control (i.e., dead) sgRNA; (2) LNP mediated delivery of GFP mRNA and gRNA for target gene 3; and (3) LNP mediated delivery of Cas9 mRNA and gRNA for target gene 3). Four mRNA concentrations were tested for each condition: 15.6 ng/ml, 62.5 ng/ml, 250 ng/ml and 1000 ng/ml. In wild-type primary mouse hepatocytes, a dose-dependent increase in insertion/deletion frequency was observed only when both Cas9 mRNA and gRNA for target gene 3 were administered. with control GFP mRNA instead of Cas9 mRNA, and the level of insertion/deletion frequency was substantially identical to those observed when Cas9 mRNA was also introduced.

[00280] An in vivo target gene 3 knockout experiment was then performed by injecting sgRNA into mice with a deleted Cas9 cassette (MAID2600) via hydrodynamic DNA delivery (HDD), lipid nanoparticle (LNP) delivery, or adeno-associated virus (AAV) injection carrying the sgRNA expression sequence by injection into the tail vein. See figures 6A-6D. As a control, the same sgRNA administration methods were paired with exogenous Cas9 expression in wild-type (WT) mice. Three groups of mice were tested for LNP mediated delivery: (1) Cas9 expressing mice (3 males + 3 females; 2 mg/kg control sgRNA + GFP mRNA); (2) Cas9 expressing mice (3 males + 3 females; 2 mg/kg sgRNA for target gene 3 + GFP mRNA); and (3) WT mice (3 males + 3 females; 2 mg/kg sgRNA for gene target 3 + Cas9 mRNA). Two groups of mice were tested for AAV-mediated delivery: (1) Cas9 expressing mice (3 males + 3 females; AAV8-sgRNA for target gene 3); and (2) mouse WT (3 male + 3 female; AAV8-sgRNA for gene target 3 + AAV8-Cas9). Two groups of mice were tested for HDD: (1) Cas9 expressing mice (3 males + 3 females; sgRNA for target gene 3); and (2) mouse WT (3 males + 3 females; sgRNA for target gene 3 + Cas9). Cas9 expressing mice exhibited consistent and significantly more targeted gene inactivation than WT mice exogenously expressing Cas9.

[00281] Unexpectedly, AAV8-mediated delivery of sgRNA target gene 3 to Cas9 expressing mice (MAID2600) was more efficient than either LNP or HDD mediated delivery when targeting liver target gene 3. See figures 6A-6D. In addition, AAV8-mediated delivery of target gene sgRNA to 3 Cas9 expressing mice was more efficient than AAV8-mediated delivery of both target gene sgRNA and Cas9 to WT mice, while no LNP-mediated delivery or HDD was observed between both conditions. significant difference shipping. See figures 6A-6D. These results indicate that AAV-mediated delivery of guide RNAs to Cas9 expressing mice may be a particularly effective tool for testing gRNA activity in vivo. Serum levels of the protein encoded by the target gene 3 (ie, target protein 3) were measured in female and male mice 7 and 21 days after administration of the CRISPR/Cas components. Mice tested included Cas9 expressing mice and wild-type mice. Controls included Cas9 expressing and WT mice that received neither sgRNA nor Cas9 nor target gene 3. Hydrodynamic delivery of guide RNA or the combination of Cas9 and guide RNA did not significantly reduce serum levels of target protein 3 compared to controls. WT mice treated with neither Cas9 nor guide RNA. For LNP-mediated delivery, administration of sgRNA to Cas9 expressing mice resulted in similar serum levels of target protein compared to WT mice administered with both Cas9 and sgRNA, and each of these conditions resulted in reduced serum levels of target protein 3 by approximately 50% compared to Cas9 expressing control mice treated with neither Cas9 nor guide RNA. However, in AAV8-mediated delivery, delivery of sgRNA to Cas9 expressing mice resulted in a several-fold decrease in serum levels of target protein 3 compared to WT mice injected with both Cas9 and sgRNA, and an even greater decrease compared to control WT mice. which were not injected with either Cas9 or guide RNA. By day 21, serum levels of target protein 3 dropped almost to the detection limit in Cas9 expressing mice injected with sgRNA via AAV8.

[00282] Figure 7 shows the percentage of NHEJ activity (indel frequency) at the target gene 3 locus in the liver of wild-type and Cas9 cassette-deleted (MAID2600) mice one month after sgRNA delivery by lipid nanoparticles (LNP) separately or together with Cas9 mRNA, hydrodynamic delivery (HDD) of sgRNA plasmid alone or together with Cas9 plasmid or AAV8-sgRNA alone or together with AAV8-Cas9. The percentage of liver cells with inserts/deletions (indels) at the locus was measured using NGS. Hydrodynamic delivery of guide RNA or a combination of Cas9 and guide RNA resulted in low indel percentages. For LNP mediated delivery, administration of sgRNA to Cas9 expressing mice resulted in a similar percentage of indels compared to WT mice administered with both Cas9 and sgRNA, and each of these conditions resulted in an indel percentage level that was approximately 40%. However, for AAV8 mediated delivery, delivery of sgRNA to Cas9 expressing mice resulted in a much higher percentage of indels (~75%) compared to WT mice injected with both Cas9 and sgRNA (~35%), and an even greater increase compared to with WT control mice treated with neither Cas9 nor guide RNA.

[00283] Further next generation sequencing (NGS) was also performed on harvested tissues. Amplicon sequencing was then used to estimate the amount of editing in collected tissues. Target specific primers are designed to produce a ~300 bp product that is slightly off-center around the intended guide cut site. "Adapter" sequences are then added to the primers, which allow individual samples to be barcoded in a secondary PCR reaction. After barcodes are added, samples are pooled and loaded into MiSeq. Five thousand to ten thousand reads are expected in the area of interest. The read matching data programs are then run to determine the exact sequence of each edit. The program then counts the number of WT (unedited) reads and provides a breakdown of the type of editing performed on all edited reads (an estimate of the number of base pairs added and/or removed in the predicted edit area).

[00284] An experiment was then performed to knock out target gene 4, which encodes a type II membrane-associated glycoprotein, by introducing Cas9 with sgRNA targeting exon 2 of target gene 4 into primary hepatocytes isolated from wild-type (WT) mice. Five different sgRNAs were tested (guides 1-5). Ribonucleoprotein Cas9/sgRNA (RNP) complexes were introduced into cells using lipofectamine. Non-homologous end joins were then assessed by next generation sequencing (NGS) to measure indel frequencies at target gene locus 4. Percent edit is a measure of the total number of NHEJ events relative to the total number of reads. NHEJ events are counted as all edits (inserts, deletions, base changes) that occur in 20 bp. before and after the incision site. The editing percentage for guides 1 to 5 was 35.4%, 37.4%, 43.8%, 51.2% and 55.8%, respectively (data not shown).

[00285] An in vivo target gene 4 knockout experiment was then performed by injecting the same five sgRNAs into individual mice with the Cas9 cassette (MAID2600) removed via AAV8. Specifically, individual guides expressed by the U6 promoter were packaged in AAV8 and administered to 6-12 week old mice with the Cas9 cassette removed by tail vein injection. The viral load administered was 1×10 ¹¹ - 1×10 ¹² , in an approximate volume of 50-100 μl. The liver was harvested 3-4 weeks after injection. Percentage editing was calculated in the same way as in primary hepatocytes and is shown in Figure 8A. The levels of editing matched and actually exceeded the levels of editing observed in primary hepatocytes. In addition, expression levels of mRNA transcribed from target gene 4 were tested. As shown in FIG. 8B, each gRNA reduced the relative levels of mRNA transcribed from target gene 4 in liver harvested 3-4 weeks post-injection.

[00286] In addition, experiments were conducted to study the percentage of editing in some other target genes in the liver. In each experiment, the mice were approximately 6-12 weeks old. For each target gene, five different guide RNAs were designed for critical exons. Guide RNAs were delivered via AAV8 by injection into the tail vein with viral loads of 1×10 ¹¹ - 1×10 ¹² in an approximate volume of 50-100 μl. The liver was harvested 3-4 weeks after injection. Percent editing was determined as explained above. The percentage of liver editing in mice with the removed Cas9 cassette (MAID2600) via AAV8-rRNA delivery is shown in Table 12. The best gRNA for each gene resulted in a 48%-70% editing in the liver in vivo.

[00287] Table 12. Percentage of editing in the liver.

Example 2 Inducibility Validation of Cas9 Expressing Mice

[00288] The LSL-Cas9 allele described in Example 1 (MAID2599) includes a loxP-site-flanked poly(A) (lox-stop-lox, or LSL) region upstream of the Cas9 coding sequence. Prior to removal of the cassette by Cre recombinase, the neomycin resistance gene will generally be efficiently transcribed and translated; however, CDS Cas9 is generally not expressed due to the presence of a strong poly(A) region that can effectively block end-to-end transcription. After removal of the neomycin cassette by Cre recombinase, the fusion mRNA for Cas9 and GFP is usually constitutively expressed by the Rosa26 promoter. This makes the Cas9 allele inducible. This is favorable for a number of reasons. The ability to edit certain genes in certain tissues (eg, immune cells) can be detrimental, while potentially triggering an immune response. In addition, under certain circumstances, mutation of a gene in the entire target individual may be lethal, while mutation of a gene in a particular tissue or cell type will be beneficial. The inducible nature of the MAID2599 allele provides greater specificity as to which tissue and cell type is edited by activating Cas9 in a tissue-specific or cell-specific manner only.

[00289] To study the inducibility of Cas9 expression in the liver in vivo, lipid nanoparticles (LNPs) containing Cre recombinase mRNA were formulated on a Precision Nanosystems Benchtop NanoAssmblr. Cre mRNA from Trilink (Cat. No. 7211) was diluted in 10 mM sodium citrate and combined through a NanoAssemblr cassette in a 3:1 ratio with a lipid combination of cationic lipid, DSPC, cholesterol and PEG-DMG in a molar ratio of 50:10:38.5 :1.5. This composition is easily absorbed by the liver. The resulting LNP-Cre was injected via the tail vein of mice with LSL-Cas9 (MAID2599) at a dose of 1 mg/kg. Control mice were not treated with LNP-Cre. Mice were sacrificed 1 week later and organs were harvested for Western blot analysis using an anti-Cas9 monoclonal antibody (7A9) (Invitrogen, cat. no. MA5-23519) and an anti-actin monoclonal antibody (C4) (Millipore Sigma, cat. no. MAB1501 ). Organs from mice with the removed Cas9 cassette (MAID2600) were used as positive controls. In these mice, the LSL cassette had already been removed by Cre recombinase. The results are shown in Figure 9, which shows experimental proof-of-concept liver-specific activation of Cas9 with LNP-Cre delivery for liver-specific gene editing.

[00290] The inducibility of Cas9-mediated gene editing was then tested in vivo. LNP-Cre was made as described above. Mice were dosed with LNP-Cre and AAV8-rRNA targeting gene 3 (co-injection via tail vein injection) in the following groups: (1) 3 LSL-Cas9 mice treated with LNP-Cre and AAV8-rRNA; (2) 3 LSL-Cas9 mice treated with LNP-Cre and PBS; (3) 3 LSL-Cas9 mice treated with PBS and AAV8-gRNA; (4) 3 LSL-Cas9 mice treated with PBS alone; (5) 3 cassette removed Cas9 mice treated with LNP-Cre; (6) 3 cassette-deleted Cas9 mice treated with AAV8-gRNA; (7) 3 cassette-deleted Cas9 mice treated with PBS; and (8) 3 WT mice (untreated). In the groups to which LNP-Cre was delivered, it was delivered at a concentration of 1 mg/kg. In groups to which AAV8-gRNA was delivered, it was delivered at a viral load of approximately 2×10 ¹¹ . Mice were bled one and three weeks after injection for serum chemistry analysis and for measurement of circulating serum target protein 3 levels. Three weeks later, tissues were also harvested for NGS and Western blot analysis. Serum levels of target protein 3 were measured by ELISA. The results are shown in Figure 10. Delivery of LNP-Cre to LSL-Cas9 mice along with AAV8 rRNA resulted in a decrease in target protein 3 serum levels, consistent with the decrease observed in cassette-deleted Cas9 mice delivered with AAV8 rRNA. These ELISA results were consistent with NGS results for percentage edits in target gene 3 in liver isolated from mice 3 weeks post-injection. See figure 11.

[00291] LSL-Cas9 mice were then bred with Jax Albumin-Cre mice (3601-Tg(Alb-cre)21Mgn; MAID3601), in which the albumin promoter is operably linked to the Cre recombinase coding sequence and drives its expression in the liver. After breeding, several tissues were collected from mice for Western blotting. Corresponding tissues were collected from LSL-Cas9 mice that were not bred to Albumin-Cre mice. Western blotting was then performed to measure Cas9 expression in the liver and brain. Actin was used as a load control. The predicted size of Cas9 was 150.48 kDa and the predicted size of actin was 41.25 kDa. 17.5 μg of liver protein lysates and brain protein lysates were used. TruCut v2 Cas9 (17.5 μg) was used as a positive control. As shown in Figure 12A, Cas9 expression was observed in the liver of LSL-Cas9/Alb-Cre mice, but not in the liver of LSL-Cas9 mice. Cas9 expression was not observed in the brain tissues of any of the mice (see Figure 12B), confirming that Cas9 expression was induced specifically in the liver.

[00292] An experiment was conducted to test Cas9-mediated gene editing in vivo in these mice. The experiment included five groups of mice aged 8-12 weeks, which were injected with AAV8-rRNA targeting gene 3 via tail vein injection: (1)3 mice LSL-Cas9:Alb-Cre (MAID2599 Het ( heterozygous), MAID3601 Het); (2) 3 mice WT:Alb-Cre (MAID2599 WT, MAID3601 Het); (3) 3 LSL-Cas9:WT mice (MAID2599 Het, MAID3601 WT); (4) 3 Cas9 mice (MAID2600 Het or Horn (homozygous)); and (5) 3 mice 75/25 (50500 WT). Mice from each group also served as controls that were not injected with AAV8-rRNA. In the groups that were delivered AAV8-rRNA, it was delivered with a viral load of approximately 2×10 ¹¹ . One week after injection, mice were bled for serum chemistry and ELISA analyses. Three weeks after injection, mice were bled and tissues were collected. The results are shown in Figure 13. Breeding LSL-Cas9 mice with Albumin-Cre mice and then injecting AAV8-rRNA resulted in a decrease in target protein 3 serum levels, consistent with the decrease seen in cassette-deleted Cas9 mice delivered AAV8-gRNA.

[00293] The mouse Cas9 expressing system described herein is capable of inducing more robust gene editing than other methods based on exogenous administration of Cas9. In addition, the system can conditionally express Cas9 based on the deletion of the neomycin cassette. By combining this system with various mouse strains in which the DNA excision is provided by Cre, the timing of Cas9-induced genome editing can be controlled and tissue-specific expression of Cas9 can be achieved in vivo.

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SEQUENCE LIST

<110>Regeneron Pharmaceuticals, Inc.

<120>CAS-EXPRESSING MOUSE EMBRYONAL STEM CELLS AND MICE

AND THEIR APPLICATIONS

<130> 57766-516569

<150>US 62/539275

<151> 2017-07-31

<160> 38

<170>PatentIn version 3.5

<210> 1

<211> 8628

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221> misc_feature

<222> (300)..(333)

<223> LoxP

<220>

<221> misc_feature

<222> (424)..(2489)

<223> Neo-PolyA

<220>

<221> misc_feature

<222> (2517)..(2550)

<223> LoxP

<220>

<221> misc_feature

<222> (2599)..(2608)

<223> Kozak

<220>

<221> misc_feature

<222> (2605)..(6777)

<223> Cas9

<220>

<221> misc_feature

<222> (2614)..(2634)

<223>Single NLS

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<223>WPRE

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<222> (8204)..(8419)

<223>bGH polyadenylation signal

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<222> (8479)..(8628)

<223>Mouse downstream Rosa26 sequence

<400> 1

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctagttgaccag 180

240

300

360

420

accatgggatcggccattgaacaagatggattgcacgcaggttctccggccgcttgggtg 480

gagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtg 540

ttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgcc 600

660

tgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaa 720

gtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatg 780

gctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaa 840

gcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggat 900

gatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcg 960

cgcatgcccgacggcgatgatctcgtcgtgacccatggcgatgcctgcttgccgaatatc 1020

1080

cgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgg 1140

gctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttc 1200

tatcgccttcttgacgagttcttctgaggggatccgctgtaagtctgcagaaattgatga 1260

1320

1380

tggggtgggattagataaatgcctgctctttactgaaggctctttactattgctttatga 1440

taatgtttcatagttggatatcataatttaaacaagcaaaaccaaattaagggccagctc 1500

1560

1620

aagaacgaga tcagcagcct ctgttccaca tacacttcat tctcagtatt gttttgccaa 1680

gttctaattc catcagaagc ttgcagatct gcgactctag aggatctgcg actctagagg 1740

atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 1800

ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 1860

gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 1920

tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 1980

tgcgactcta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa 2040

aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 2100

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 2160

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 2220

atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat ttgtagaggt 2280

tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc 2340

aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 2400

cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 2460

catcaatgta tcttatcatg tctggatccc catcaagctg atcgggaacc cttaatataa 2520

cttcgtataa tgtatgctat acgaagttat taggtccctc gacctgcagc ccaagctagt 2580

gcccgggaat tcgctagggc caccatggac aagcccaaga aaaagcggaa agtgaagtac 2640

agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac 2700

aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg acaggcacag catcaagaag 2760

aacctgatcg gcgccctgct gttcgacagc ggcgaaacag ccgaggccac cagactgaag 2820

agaaccgcca gaagaagata caccaggcgg aagaacagga tctgctatct gcaagagatc 2880

ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc 2940

ctggtggaag aggacaagaa gcacgagaga caccccatct tcggcaacat cgtggacgag 3000

gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc 3060

accgacaagg ccgacctgag actgatctac ctggccctgg cccacatgat caagttcaga 3120

ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc 3180

atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc 3240

gtggacgcca aggctatcct gtctgccaga ctgagcaaga gcagaaggct ggaaaatctg 3300

atcgcccagc tgcccggcga gaagaagaac ggcctgttcg gcaacctgat tgccctgagc 3360

ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag 3420

ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag 3480

tacgccgacc tgttcctggc cgccaagaac ctgtctgacg ccatcctgct gagcgacatc 3540

ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac 3600

gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag 3660

aagtacaaag aaatcttctt cgaccagagc aagaacggct acgccggcta catcgatggc 3720

ggcgctagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc 3780

accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgagaaagca gagaaccttc 3840

3900

3960

ttcaggatcc cctactacgt gggccccctg gccagaggca acagcagatt cgcctggatg 4020

accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc 4080

gccagcgccc agagcttcat cgagagaatg acaaacttcg ataagaacct gcccaacgag 4140

aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtacaa cgagctgacc 4200

aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa 4260

aaggccatcg tggacctgct gttcaagacc aacagaaaag tgaccgtgaa gcagctgaaa 4320

gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat 4380

agattcaacg ccctccctgggg cacataccac gatctgctga aaattatcaa ggacaaggac 4440

ttcctggata acgaagagaa cgaggacatt ctggaagata tcgtgctgac cctgacactg 4500

tttgaggacc gcgagatgat cgaggaaagg ctgaaaacct acgctcacct gttcgacgac 4560

aaagtgatga agcagctgaa gagaaggcgg tacaccggct ggggcaggct gagcagaaag 4620

ctgatcaacg gcatcagaga caagcagagc ggcaagacaa tcctggattt cctgaagtcc 4680

gacggcttcg ccaaccggaa cttcatgcag ctgatccacg acgacagcct gacattcaaa 4740

gaggacatcc agaaagccca ggtgtccggc cagggcgact ctctgcacga gcatatcgct 4800

aacctggccg gcagccccgc tatcaagaag ggcatcctgc agacagtgaa ggtggtggac 4860

gagctcgtga aagtgatggg cagacacaag cccgagaaca tcgtgatcga gatggctaga 4920

gagaaccaga ccacccagaa gggacagaag aactcccgcg agaggatgaa gagaatcgaa 4980

gagggcatca aaagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag 5040

ctgcagaacg agaagctgta cctgtactac ctgcagaatg gccgggatat gtacgtggac 5100

caggaactgg acatcaacag actgtccgac tacgatgtgg accatatcgt gcctcagagc 5160

tttctgaagg acgactccat cgataacaaa gtgctgactc ggagcgacaa gaacagaggc 5220

aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcgacag 5280

ctgctgaacg ccaagctgat tacccagagg aagttcgata acctgaccaa ggccgagaga 5340

ggcggcctga gcgagctgga taaggccggc ttcatcaaga ggcagctggt ggaaaccaga 5400

cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgaa 5460

aacgataagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat 5520

ttcgggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac 5580

gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa 5640

agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc 5700

gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt 5760

ttcaagaccg aaatcaccct ggccaacggc gagatcagaa agcgccctct gatcgagaca 5820

aacggcgaaa ccggggagat cgtgtgggat aagggcagag acttcgccac agtgcgaaag 5880

gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc 5940

agcaaagagt ctatcctgcc caagaggaac agcgacaagc tgatcgccag aaagaaggac 6000

tgggacccca agaagtacgg cggcttcgac agccctaccg tggcctactc tgtgctggtg 6060

gtggctaagg tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg 6120

atcaccatca tggaaagaag cagctttgag aagaacccta tcgactttct ggaagccaag 6180

ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag 6240

ctggaaaacg gcagaaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgag 6300

6360

6420

ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgcc 6480

aatctggaca aggtgctgtc tgcctacaac aagcacaggg acaagcctat cagagagcag 6540

gccgagaata tcatccacct gttcaccctg acaaacctgg gcgctcctgc cgccttcaag 6600

tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc 6660

accctgatcc accagagcat caccggcctg tacgagacaa gaatcgacct gtctcagctg 6720

ggaggcgaca agagacctgc cgccactaag aaggccggac aggccaaaaa gaagaaggga 6780

agcggagcca ctaacttctc cctgttgaaa caagcagggg atgtcgaaga gaatcccgggg 6840

ccagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 6900

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 6960

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 7020

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 7080

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 7140

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 7200

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 7260

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 7320

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 7380

gacctacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 7440

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 7500

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 7560

acgcgtatgc atggccggcc ctgcaggaat tcgatatcaa gcttatcgat aatcaacctc 7620

tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 7680

tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 7740

ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 7800

tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 7860

ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 7920

cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 7980

acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 8040

ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 8100

accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 8160

ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgcgacctc gacctcgact 8220

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 8280

gaaggtgcca ctcccactgt ccttttcctaa taaaatgagg aaattgcatc gcattgtctg 8340

agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 8400

gaagacaatg gcaggcatgc tggggaacta gtggtgccag ggcgtgccct tgggctcccc 8460

gggcgcggcg gccatcgctc gagtaaaatt ggagggacaa gacttcccac agattttcgg 8520

ttttgtcggg aagttttttta ataggggcaa ataaggaaaa tgggaggata ggtagtcatc 8580

tggggtttta tgcagcaaaa ctacaggtta ttattgcttg tgatccgc 8628

<210> 2

<211> 18

<212> PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 2

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

GlyPro

<210> 3

<211> 19

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 3

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

ProGlyPro

<210> 4

<211> 20

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 4

GlnCysThrAsnTyrAlaLeuLeuLysLeuAlaGlyAspValGluSer

1 5 10 15

AsnProGlyPro

twenty

<210> 5

<211> 22

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 5

ValLysGlnThrLeuAsnPheAspLeuLeuLysLeuAlaGlyAspVal

1 5 10 15

Glu Ser Asn Pro Gly Pro

twenty

<210> 6

<211> 82

<212>RNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 6

guuggaacca uucaaaacag cauagcaagu uaaaauaagg cuaguccguu aucaacuuga 60

aaaagggcaccgagucggugc 82

<210> 7

<211> 76

<212>RNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 7

60

ggcaccgagucggugc 76

<210> 8

<211> 86

<212>RNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 8

60

uugaaaaaguggcaccgagucggugc 86

<210> 9

<211> 23

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> misc_feature

<222> (2)..(21)

<223> n = A, T, C or G

<400> 9

gnnnnnnnnnnnnnnnnnnngg 23

<210> 10

<211> 23

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> misc_feature

<222> (1)..(21)

<223> n = A, T, C or G

<400> 10

nnnnnnnnnnnnnnnnnnnngg 23

<210> 11

<211> 25

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> misc_feature

<222> (3)..(23)

<223> n = A, T, C or G

<400> 11

ggnnnnnnnnnnnnnnnnnnnngg 25

<210> 12

<211> 6411

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221> misc_feature

<222> (300)..(333)

<223> LoxP

<220>

<221> misc_feature

<222> (382)..(391)

<223> Kozak

<220>

<221> misc_feature

<222> (388)..(4560)

<223> Cas9

<220>

<221> misc_feature

<222> (397)..(417)

<223>Single NLS

<220>

<221> misc_feature

<222> (4513)..(4560)

<223>Double NLS

<220>

<221> misc_feature

<222> (4561)..(4626)

<223> P2A

<220>

<221> misc_feature

<222> (4627)..(5340)

<223> eGFP

<220>

<221> misc_feature

<222> (5390)..(5986)

<223>WPRE

<220>

<221> misc_feature

<222> (5987)..(6202)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (6262)..(6411)

<223>Mouse downstream Rosa26 sequence

<400> 12

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctagttgaccag 180

240

300

taacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagct 360

420

tacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgag 480

540

aagaacctgatcggcgccctgctgttcgacagcggcgaaacagccgaggccaccagactg 600

660

atcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtcc 720

ttcctggtggaagaggacaagaagcacgagagacaccccatcttcggcaacatcgtggac 780

gaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggac 840

agcaccgacaaggccgacctgagactgatctacctggccctggcccacatgatcaagttc 900

agaggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctg 960

ttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc 1020

ggcgtggacgccaaggctatcctgtctgccagactgagcaagagcagaaggctggaaaat 1080

1140

agcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactg 1200

1260

cagtacgccgacctgttcctggccgccaagaacctgtctgacgccatcctgctgagcgac 1320

1380

tacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcct 1440

gagaagtacaaagaaatcttcttcgaccagagcaagaacggctacgccggctacatcgat 1500

ggcggcgctagcgggaagagttctacaagttcatcaagcccatcctggaaaagatggac 1560

ggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgagaaagcagagaacc 1620

ttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgctatcctgaga 1680

1740

accttcaggatcccctactacgtgggccccctggccagaggcaacagcagattcgcctgg 1800

1860

ggcgccagcgcccagagcttcatcgagagaatgacaaacttcgataagaacctgcccaac 1920

gagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtacaacgagctg 1980

2040

aaaaaggccatcgtggacctgctgttcaagaccaacagaaaagtgaccgtgaagcagctg 2100

2160

2220

gacttcctggataacgaagagaacgaggacattctggaagatatcgtgctgaccctgaca 2280

2340

gacaaagtgatgaagcagctgaagagaaggcggtacaccggctggggcaggctgagcaga 2400

2460

2520

aaagaggacatccagaaagcccaggtgtccggccagggcgactctctgcacgagcatatc 2580

gctaacctggccggcagccccgctatcaagaagggcatcctgcagacagtgaaggtggtg 2640

2700

agagagaaccagaccacccagaagggacagaagaactcccgcgagaggatgaagagaatc 2760

gaagagggcatcaaagagctgggcagccagatcctgaaagaacacccgtggaaaacacc 2820

cagctgcagaacgagaagctgtacctgtactacctgcagaatggccgggatatgtacgtg 2880

gaccaggaactggacatcaacagactgtccgactacgatgtggaccatatcgtgcctcag 2940

agctttctgaaggacgactccatcgataacaaagtgctgactcggagcgacaagaacaga 3000

ggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagaagatgaagaactactggcga 3060

3120

agaggcggcctgagcgagctggataaggccggcttcatcaagaggcagctggtggaaacc 3180

agacagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgac 3240

gaaaacgataagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtcc 3300

gatttccgggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcc 3360

3420

3480

3540

tttttcaagaccgaaatcaccctggccaacggcgagatcagaaagcgccctctgatcgag 3600

acaaacggcgaaaccggggagatcgtgtgggataagggcagagacttcgccacagtgcga 3660

aaggtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggc 3720

3780

3840

3900

gggatcaccatcatggaaagaagcagctttgagaagaaccctatcgactttctggaagcc 3960

aagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttc 4020

gagctggaaaacggcagaaagagaatgctggcctctgccggcgaactgcagaagggaaac 4080

4140

4200

4260

4320

caggccgagaatatcatccacctgttcaccctgacaaacctgggcgctcctgccgccttc 4380

aagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggac 4440

gccaccctgatccaccagagcatcaccggcctgtacgagacaagaatcgacctgtctcag 4500

4560

ggaagcggagcactaacttctccctgttgaaacaagcaggggatgtcgaagagaatccc 4620

gggccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg 4680

4740

tacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc 4800

accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatg 4860

aagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatc 4920

4980

ctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctgggg 5040

cacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaag 5100

aacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctc 5160

gccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaac 5220

cactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatg 5280

gtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag 5340

taaacgcgtatgcatggccggccctgcaggaattcgatatcaagcttatcgataatcaac 5400

ctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttta 5460

cgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctt 5520

5580

ttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttgggg 5640

gcattgccaccacctgtcagctccttttccgggactttcgctttccccctccctattgcca 5700

5760

5820

ttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccag 5880

cggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttc 5940

gccctcagacgagtcggatctccctttgggccgcctccccgcatcgcgacctcgacctcg 6000

actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgacc 6060

6120

ctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggat 6180

tgggaagacaatggcaggcatgctggggaactagtggtgccagggcgtgcccttgggctc 6240

6300

6360

atctggggttttatgcagcaaaactacaggttattattgcttgtgatccgc 6411

<210> 13

<211> 1651

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>MISC_FEATURE

<222> (1)..(1391)

<223> Cas9

<220>

<221> MISC_FEATURE

<222> (1392)..(1413)

<223> P2A

<220>

<221> MISC_FEATURE

<222> (1414)..(1651)

<223> eGFP

<400> 13

Met Asp Lys Pro Lys Lys Lys Arg Lys Val Lys Tyr Ser Ile Gly Leu

1 5 10 15

Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr

20 25 30

Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His

35 40 45

Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu

50 55 60

Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr

65 70 75 80

Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu

85 90 95

Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe

100 105 110

Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn

115 120 125

Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His

130 135 140

Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu

145 150 155 160

Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu

165 170 175

Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe

180 185 190

Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile

195 200 205

Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser

210 215 220

Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys

225 230 235 240

Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr

245 250 255

Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln

260 265 270

Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln

275 280 285

Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser

290 295 300

Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr

305 310 315 320

Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His

325 330 335

Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu

340 345 350

Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly

355 360 365

Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys

370 375 380

Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu

385 390 395 400

Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser

405 410 415

Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg

420 425 430

Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu

435 440 445

Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg

450 455 460

Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile

465 470 475 480

Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln

485 490 495

Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu

500 505 510

Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr

515 520 525

Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro

530 535 540

Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe

545 550 555 560

Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe

565 570 575

Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp

580 585 590

Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile

595 600 605

Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu

610 615 620

Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu

625 630 635 640

Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys

645 650 655

Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys

660 665 670

Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp

675 680 685

Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile

690 695 700

His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val

705 710 715 720

Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly

725 730 735

Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp

740 745 750

Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile

755 760 765

Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser

770 775 780

Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

785 790 795 800

Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu

805 810 815

Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp

820 825 830

Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile

835 840 845

Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu

850 855 860

Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu

865 870 875 880

Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala

885 890 895

Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg

900 905 910

Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu

915 920 925

Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser

930 935 940

Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val

945 950 955 960

Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp

965 970 975

Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His

980 985 990

Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr

995 1000 1005

Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr

1010 1015 1020

Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys

1025 1030 1035

Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe

1040 1045 1050

Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro

1055 1060 1065

Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys

1070 1075 1080

Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln

1085 1090 1095

Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser

1100 1105 1110

Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1115 1120 1125

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1130 1135 1140

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys

1145 1150 1155

Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile

1160 1165 1170

Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe

1175 1180 1185

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile

1190 1195 1200

Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys

1205 1210 1215

Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu

1220 1225 1230

Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His

1235 1240 1245

Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln

1250 1255 1260

Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu

1265 1270 1275

Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn

1280 1285 1290

Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro

1295 1300 1305

Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr

1310 1315 1320

Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile

1325 1330 1335

Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr

1340 1345 1350

Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1355 1360 1365

Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys

1370 1375 1380

Ala Gly Gln Ala Lys Lys Lys Lys Gly Ser Gly Ala Thr Asn Phe

1385 1390 1395

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

1400 1405 1410

Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1415 1420 1425

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser

1430 1435 1440

Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys

1445 1450 1455

Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu

1460 1465 1470

Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro

1475 1480 1485

Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu

1490 1495 1500

Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn

1505 1510 1515

Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val

1520 1525 1530

Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn

1535 1540 1545

Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val

1550 1555 1560

Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe

1565 1570 1575

Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp

1580 1585 1590

His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu

1595 1600 1605

Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp

1610 1615 1620

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr

1625 1630 1635

Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

1640 1645 1650

<210> 14

<211> 10439

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221>misc_feature

<222> (195)..(1913)

<223>CAGG promoter

<220>

<221> misc_feature

<222> (1996)..(2029)

<223> LoxP

<220>

<221> misc_feature

<222> (2120)..(4185)

<223> Neo-PolyA

<220>

<221> misc_feature

<222> (4213)..(4246)

<223> LoxP

<220>

<221> misc_feature

<222> (4341)..(4350)

<223> Kozak

<220>

<221> misc_feature

<222> (4350)..(4415)

<223> 3xFLAG

<220>

<221> misc_feature

<222> (4416)..(8588)

<223> Cas9

<220>

<221> misc_feature

<222> (4425)..(4445)

<223>Single NLS

<220>

<221> misc_feature

<222> (8541)..(8588)

<223>Double NLS

<220>

<221> misc_feature

<222> (8589)..(8654)

<223> P2A

<220>

<221> misc_feature

<222> (8655)..(9368)

<223> eGFP

<220>

<221> misc_feature

<222> (9418)..(10014)

<223>WPRE

<220>

<221> misc_feature

<222> (10015)..(10230)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (10290)..(10439)

<223>Mouse downstream Rosa26 sequence

<400> 14

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctccttaattaa 180

ggcctccaaggcctactagttattaatagtaatcaattacggggtcattagttcatagcc 240

catatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgccca 300

acgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaataggga 360

ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc 420

aagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcct 480

ggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtat 540

tagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatct 600

660

tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg 720

gcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttc 780

840

ggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgccc 900

gccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcc 960

1020

aagccttgaggggctccgggaggggccctttgtgcggggggagcggctcggggggtgcgtg 1080

1140

1200

1260

1320

cccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcg 1380

1440

ggggccgcctcggccggggagggctcgggggaggggcgcggcggcccccggagcgccgg 1500

1560

gcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcac 1620

1680

gggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtcc 1740

gcggggggacggctgccttcggggggggacggggcagggcggggttcggcttctggcgtgt 1800

gaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagct 1860

cctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattcgctaggag 1920

1980

2040

acctgcaggaattgttgacaattaatcatcggcatagtatatcggcatagtataatacga 2100

2160

tccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctg 2220

2280

cgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggc 2340

2400

gctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccga 2460

gaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctg 2520

2580

tcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgtt 2640

cgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatggcgatgc 2700

ctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccg 2760

2820

gcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattc 2880

gcagcgcatcgccttctatcgccttcttgacgagttcttctgaggggatccgctgtaagt 2940

3000

3060

gctacggggggtgggggtggggtgggattagataaatgcctgctctttactgaaggctctt 3120

3180

aattaagggccagctcattcctcccactcatgatctatagatctatagatctctcgtggg 3240

atcattgttttctcttgattcccactttgtggttctaagtactgtggtttccaaatgtg 3300

tcagtttcatagcctgaagaacgagatcagcagcctctgttccacatacacttcattctc 3360

3420

tctgcgactc taatcagcca taccacattt gtagaggttt tacttgcttt 3480

aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt 3540

taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 3600

aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 3660

ttatcatgtc tggatctgcg actctagagg atcataatca gccataccac atttgtagag 3720

3780

gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc 3840

3900

ctcatcaatg tatcttatca tgtctggatc tgcgactcta gaggatcata atcagccata 3960

ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 4020

aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 4080

aataaagcaa tagcatcaca aatttcacaa ataaagcatt ttttcactg cattctagtt 4140

gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccccatc aagctgatcc 4200

ggaaccctta atataacttc gtataatgta tgctatacga agttattagg tccctcgacc 4260

tgcagcccaa gctagtgccc gggtaggtcc ctcgacctgc agcccaagct agatcgaatt 4320

cggccggcct tcgaacacgt gccaccatgg actataagga ccacgacgga gactacaagg 4380

4440

aagtgaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca 4500

4560

gcatcaagaa gaacctgatc ggcgccctgc tgttcgacag cggcgaaaca gccgaggcca 4620

ccagactgaa gagaaccgcc agaagaagat acaccaggcg gaagaacagg atctgctatc 4680

tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg 4740

aagagtcctt cctggtggaa gaggacaaga agcacgagag acaccccatc ttcggcaaca 4800

tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac 4860

tggtggacag caccgacaag gccgacctga gactgatcta cctggccctg gcccacatga 4920

tcaagttcag aggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg 4980

acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca 5040

acgccagcgg cgtggacgcc aaggctatcc tgtctgccag actgagcaag agcagaaggc 5100

tggaaaatct gatcgcccag ctgcccggcg agaagaagaa cggcctgttc ggcaacctga 5160

ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg 5220

ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga 5280

tcggcgacca gtacgccgac ctgttcctgg ccgccaagaa cctgtctgac gccatcctgc 5340

tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga 5400

tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc 5460

agctgcctga gaagtacaaa gaaatcttct tcgaccagag caagaacggc tacgccggct 5520

acatcgatgg cggcgctagc caggaagagt tctacaagtt catcaagccc atcctggaaa 5580

agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgagaaagc 5640

agagaacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcta 5700

tcctgagaag gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga 5760

agatcctgac cttcaggatc ccctactacg tgggccccct ggccagaggc aacagcagat 5820

tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg 5880

tggacaaggg cgccagcgcc cagagcttca tcgagagaat gacaaacttc gataagaacc 5940

tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtaca 6000

acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg 6060

gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caacagaaaa gtgaccgtga 6120

agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg 6180

gcgtggaaga tagattcaac gcctccctgg gcacatacca cgatctgctg aaaattatca 6240

aggacaagga cttcctggat aacgaagaga acgaggacat tctggaagat atcgtgctga 6300

ccctgacact gtttgaggac cgcgagatga tcgaggaaag gctgaaaacc tacgctcacc 6360

tgttcgacga caaagtgatg aagcagctga agagaaggcg gtacaccggc tggggcaggc 6420

tgagcagaaa gctgatcaac ggcatcagag acaagcagag cggcaagaca atcctggatt 6480

tcctgaagtc cgacggcttc gccaaccgga acttcatgca gctgatccac gacgacagcc 6540

tgacattcaa agaggacatc cagaaagccc aggtgtccgg ccagggcgac tctctgcacg 6600

agcatatcgc taacctggcc ggcagccccg ctatcaagaa gggcatcctg cagacagtga 6660

aggtggtgga cgagctcgtg aaagtgatgg gcagacacaa gcccgagaac atcgtgatcg 6720

agatggctag agagaaccag accacccaga agggacagaa gaactcccgc gagaggatga 6780

agagaatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg 6840

aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat ggccgggata 6900

6960

tgcctcagag ctttctgaag gacgactcca tcgataacaa agtgctgact cggagcgaca 7020

agaacagagg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact 7080

actggcgaca gctgctgaac gccaagctga ttacccagag gaagttcgat aacctgacca 7140

aggccgagag aggcggcctg agcgagctgg ataaggccgg cttcatcaag aggcagctgg 7200

tggaaaccag acagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta 7260

agtacgacga aaacgataag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc 7320

tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc 7380

accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc 7440

ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga 7500

tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca 7560

tcatgaactt tttcaagacc gaaatcaccc tggccaacgg cgagatcaga aagcgccctc 7620

tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggcaga gacttcgcca 7680

cagtgcgaaa ggtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga 7740

caggcggctt cagcaaagag tctatcctgc ccaaggaa cagcgacaag ctgatcgcca 7800

gaaagaagga ctgggacccc aagaagtacg gcggcttcga cagccctacc gtggcctact 7860

ctgtgctggt ggtggctaag gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag 7920

agctgctggg gatcaccatc atggaaagaa gcagctttga gaagaaccct atcgactttc 7980

tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact 8040

ccctgttcga gctggaaaac ggcagaaaga gaatgctggc ctctgccggc gaactgcaga 8100

agggaaacga gctggccctg cctagcaaat atgtgaactt cctgtacctg gcctcccact 8160

8220

ataagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc 8280

tggccgacgc caatctggac aaggtgctgt ctgcctacaa caagcacagg gacaagccta 8340

tcagagagca ggccgagaat atcatccacc tgttcaccct gacaaacctg ggcgctcctg 8400

ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg 8460

tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca agaatcgacc 8520

tgtctcagct gggaggcgac aagagacctg ccgccactaa gaaggccgga caggccaaaa 8580

agaagaaggg aagcggagcc actaacttct ccctgttgaa acaagcaggg gatgtcgaag 8640

agaatcccgg gccagtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 8700

tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 8760

atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 8820

cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 8880

accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 8940

gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 9000

gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 9060

tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 9120

agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 9180

tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 9240

ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 9300

atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 9360

tgtacaagta aacgcgtatg catggccggc cctgcaggaa ttcgatatca agcttatcga 9420

taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 9480

tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 9540

tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 9600

gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 9660

tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 9720

tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 9780

gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 9840

cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 9900

caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 9960

tcgccttcgc cctcagacga gtcggatctc ccttttgggcc gcctccccgc atcgcgacct 10020

cgacctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 10080

ccttgaccct ggaaggtgcc actcccactg tccttttccta ataaaatgag gaaattgcat 10140

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 10200

gggaggattg ggaagacaat ggcaggcatg ctggggaact agtggtgcca gggcgtgccc 10260

ttgggctccc cgggcgcggc ggccatcgct cgagtaaaat tggagggaca agacttccca 10320

cagattttcg gttttgtcgg gaagtttttt aataggggca aataaggaaa atgggaggat 10380

aggtagtcat ctggggtttt atgcagcaaa actacaggtt attattgctt gtgatccgc 10439

<210> 15

<211> 8222

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221>misc_feature

<222> (195)..(1913)

<223>CAGG promoter

<220>

<221> misc_feature

<222> (1996)..(2029)

<223> LoxP

<220>

<221> misc_feature

<222> (2124)..(2133)

<223> Kozak

<220>

<221> misc_feature

<222> (2133)..(2198)

<223> 3xFLAG

<220>

<221> misc_feature

<222> (2199)..(6371)

<223> Cas9

<220>

<221> misc_feature

<222> (2208)..(2228)

<223>Single NLS

<220>

<221> misc_feature

<222> (6324)..(6371)

<223>Double NLS

<220>

<221> misc_feature

<222> (6372)..(6437)

<223> P2A

<220>

<221> misc_feature

<222> (6438)..(7151)

<223> eGFP

<220>

<221> misc_feature

<222> (7201)..(7797)

<223>WPRE

<220>

<221> misc_feature

<222> (7798)..(8013)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (8073)..(8222)

<223>Mouse downstream Rosa26 sequence

<400> 15

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctccttaattaa 180

ggcctccaaggcctactagttattaatagtaatcaattacggggtcattagttcatagcc 240

catatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgccca 300

acgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaataggga 360

ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc 420

aagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcct 480

ggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtat 540

tagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatct 600

660

tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg 720

gcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttc 780

840

ggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgccc 900

gccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcc 960

1020

aagccttgaggggctccgggaggggccctttgtgcggggggagcggctcggggggtgcgtg 1080

1140

1200

1260

1320

cccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcg 1380

1440

ggggccgcctcggccggggagggctcgggggaggggcgcggcggcccccggagcgccgg 1500

1560

gcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcac 1620

1680

gggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtcc 1740

gcggggggacggctgccttcggggggggacggggcagggcggggttcggcttctggcgtgt 1800

gaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagct 1860

cctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattcgctaggag 1920

1980

2040

acctgcagcccaagctagtgcccgggtaggtccctcgacctgcagcccaagctagatcga 2100

attcggccggccttcgaacacgtgccaccatggactataaggacccgacggagactaca 2160

2220

ggaaagtgaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtga 2280

2340

acagcatcaagaagaacctgatcggcgccctgctgttcgacagcggcgaaacagccgagg 2400

ccaccagactgaagagaaccgccagaagaagatacaccaggcggaagaacaggatctgct 2460

2520

tggaagagtccttcctggtggaagaggacaagaagcacgagagacaccccatcttcggca 2580

acatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaaga 2640

aactggtggacagcaccgacaaggccgacctgagactgatctacctggccctggcccaca 2700

2760

tggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaacccca 2820

2880

ggctggaaaatctgatcgcccagctgcccggcgagaagaagaagaacggcctgttcggcaacc 2940

tgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgagg 3000

3060

agatcggcgaccagtacgccgacctgttcctggccgccaagaacctgtctgacgccatcc 3120

3180

tgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggc 3240

agcagctgcctgagaagtacaaagaaatcttcttcgaccagagcaagaacggctacgccg 3300

3360

3420

agcagagaaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacg 3480

3540

agaagatcctgaccttcaggatcccctactacgtgggccccctggccagaggcaacagca 3600

3660

3720

3780

acaacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctga 3840

gcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaacagaaaagtgaccg 3900

tgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatct 3960

ccggcgtggaagatagattcaacgcctccctgggcacataccacgatctgctgaaaatta 4020

4080

tgaccctgacactgtttgaggaccgcgagatgatcgaggaaaggctgaaaacctacgctc 4140

acctgttcgacgacaaagtgatgaagcagctgaagagaaggcggtacaccggctggggca 4200

ggctgagcagaaagctgatcaacggcatcagagacaagcagagcggcaagacaatcctgg 4260

atttcctgaagtccgacggcttcgccaaccggaacttcatgcagctgatccacgacgaca 4320

4380

acgagcatatcgctaacctggccggcagccccgctatcaagaagggcatcctgcagacag 4440

tgaaggtggtggacgagctcgtgaaagtgatgggcagacacaagcccgagaacatcgtga 4500

4560

tgaagagaatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacacccg 4620

tggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatggccggg 4680

atatgtacgtggaccaggaactggacatcaacagactgtccgactacgatgtggaccata 4740

4800

acaagaacagaggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaaga 4860

actactggcgacagctgctgaacgccaagctgattacccagaggaagttcgataacctga 4920

ccaaggccgagagaggcggcctgagcgagctggataaggccggcttcatcaagaggcagc 4980

5040

ctaagtacgacgaaaacgataagctgatccgggaagtgaaagtgatcaccctgaagtcca 5100

5160

accaccgccccgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagt 5220

5280

tgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagca 5340

acatcatgaactttttcaagaccgaaatcaccctggccaacggcgagatcagaaagcgcc 5400

5460

ccacagtgcgaaaggtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgc 5520

5580

ccagaaagaaggactgggaccccaagaagtacggcggcttcgacagccctaccgtggcct 5640

actctgtgctggtggtggctaaggtggaaaagggcaagtccaagaaactgaagagtgtga 5700

aagagctgctggggatcaccatcatggaaagaagcagctttgagaagaaccctatcgact 5760

ttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagt 5820

actccctgttcgagctggaaaacggcagaaagagaatgctggcctctgccggcgaactgc 5880

agaagggaaacgagctggccctgcctagcaaatatgtgaacttcctgtacctggcctccc 5940

actatgagaagctgaagggcagccctgaggacaacgaacagaaacagctgtttgtggaac 6000

6060

6120

ctatcagagagcaggccgagaatatcatccacctgttcaccctgacaaacctgggcgctc 6180

ctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaag 6240

aggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacaagaatcg 6300

acctgtctcagctgggaggcgacaagagacctgccgccactaagaaggccggacagcca 6360

6420

aagagaatcccggggccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcc 6480

tggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagg 6540

gcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccg 6600

tgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctacc 6660

6720

6780

agggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggca 6840

acatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccg 6900

acaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggca 6960

gcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgc 7020

tgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagc 7080

gcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacg 7140

agctgtacaagtaaacgcgtatgcatggccggccctgcaggaattcgatatcaagcttat 7200

cgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgt 7260

tgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttc 7320

ccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgagga 7380

7440

cactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccct 7500

7560

7620

gctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggc 7680

cctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcg 7740

tcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgcga 7800

7860

7920

catcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagca 7980

aggggggattgggaagacaatggcaggcatgctggggaactagtggtgccagggcgtg 8040

8100

8160

gataggtagtcatctggggttttatgcagcaaaactacacaggttattattgcttgtgatcc 8220

gc 8222

<210> 16

<211> 1673

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(22)

<223> 3xFLAG

<220>

<221> MISC_FEATURE

<222> (23)..(1413)

<223> Cas9

<220>

<221> MISC_FEATURE

<222> (1414)..(1435)

<223> P2A

<220>

<221> MISC_FEATURE

<222> (1436)..(1673)

<223> eGFP

<400> 16

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys Met Asp Lys Pro Lys Lys Lys Arg Lys Val

20 25 30

Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala

35 40 45

Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu

50 55 60

Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu

65 70 75 80

Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr

85 90 95

Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln

100 105 110

Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His

115 120 125

Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg

130 135 140

His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys

145 150 155 160

Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp

165 170 175

Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys

180 185 190

Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser

195 200 205

Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu

210 215 220

Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile

225 230 235 240

Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala

245 250 255

Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala

260 265 270

Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala

275 280 285

Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu

290 295 300

Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu

305 310 315 320

Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg

325 330 335

Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys

340 345 350

Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val

355 360 365

Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser

370 375 380

Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu

385 390 395 400

Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu

405 410 415

Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg

420 425 430

Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu

435 440 445

His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp

450 455 460

Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr

465 470 475 480

Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg

485 490 495

Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp

500 505 510

Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp

515 520 525

Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr

530 535 540

Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr

545 550 555 560

Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala

565 570 575

Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln

580 585 590

Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu

595 600 605

Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His

610 615 620

Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu

625 630 635 640

Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu

645 650 655

Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe

660 665 670

Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp

675 680 685

Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser

690 695 700

Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg

705 710 715 720

Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp

725 730 735

Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His

740 745 750

Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln

755 760 765

Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys

770 775 780

Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln

785 790 795 800

Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly

805 810 815

Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn

820 825 830

Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly

835 840 845

Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp

850 855 860

Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser

865 870 875 880

Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser

885 890 895

Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

900 905 910

Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn

915 920 925

Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly

930 935 940

Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val

945 950 955 960

Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp

965 970 975

Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val

980 985 990

Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn

995 1000 1005

Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly

1010 1015 1020

Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

1025 1030 1035

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1040 1045 1050

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr

1055 1060 1065

Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn

1070 1075 1080

Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1085 1090 1095

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg

1100 1105 1110

Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu

1115 1120 1125

Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg

1130 1135 1140

Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys

1145 1150 1155

Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu

1160 1165 1170

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser

1175 1180 1185

Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe

1190 1195 1200

Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu

1205 1210 1215

Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe

1220 1225 1230

Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu

1235 1240 1245

Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn

1250 1255 1260

Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1265 1270 1275

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1280 1285 1290

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg

1295 1300 1305

Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr

1310 1315 1320

Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1325 1330 1335

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe

1340 1345 1350

Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr

1355 1360 1365

Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly

1370 1375 1380

Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys

1385 1390 1395

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1400 1405 1410

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp

1415 1420 1425

Val Glu Glu Asn Pro Gly Pro Val Ser Lys Gly Glu Glu Leu Phe

1430 1435 1440

Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn

1445 1450 1455

Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr

1460 1465 1470

Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu

1475 1480 1485

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val

1490 1495 1500

Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe

1505 1510 1515

Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

1520 1525 1530

Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

1535 1540 1545

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

1550 1555 1560

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

1565 1570 1575

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys

1580 1585 1590

Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp

1595 1600 1605

Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile

1610 1615 1620

Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr

1625 1630 1635

Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met

1640 1645 1650

Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met

1655 1660 1665

Asp Glu Leu Tyr Lys

1670

<210> 17

<211> 7207

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221>misc_feature

<222> (300)..(333)

<223> LoxP

<220>

<221> misc_feature

<222> (424)..(2489)

<223> Neo-PolyA

<220>

<221> misc_feature

<222> (2517)..(2550)

<223> LoxP

<220>

<221> misc_feature

<222> (2599)..(2608)

<223> Kozak

<220>

<221> misc_feature

<222> (2605)..(6777)

<223> Cas9

<220>

<221> misc_feature

<222> (2614)..(2634)

<223>Single NLS

<220>

<221> misc_feature

<222> (6730)..(6777)

<223>Double NLS

<220>

<221> misc_feature

<222> (6783)..(6998)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (7058)..(7207)

<223>Mouse downstream Rosa26 sequence

<400> 17

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctagttgaccag 180

240

300

360

420

accatgggatcggccattgaacaagatggattgcacgcaggttctccggccgcttgggtg 480

gagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtg 540

ttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgcc 600

ctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttcct 660

tgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaa 720

gtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatg 780

gctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaa 840

gcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggat 900

gatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcg 960

cgcatgcccgacggcgatgatctcgtcgtgacccatggcgatgcctgcttgccgaatatc 1020

1080

1140

gctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttc 1200

tatcgccttcttgacgagttcttctgaggggatccgctgtaagtctgcagaaattgatga 1260

1320

1380

tggggtgggattagataaatgcctgctctttactgaaggctctttactattgctttatga 1440

taatgtttcatagttggatatcataatttaaacaagcaaaaccaaattaagggccagctc 1500

1560

1620

aagaacgaga tcagcagcct ctgttccaca tacacttcat tctcagtatt gttttgccaa 1680

gttctaattc catcagaagc ttgcagatct gcgactctag aggatctgcg actctagagg 1740

atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 1800

ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 1860

gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 1920

tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 1980

tgcgactcta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa 2040

aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 2100

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 2160

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 2220

atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat ttgtagaggt 2280

tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc 2340

aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 2400

cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 2460

catcaatgta tcttatcatg tctggatccc catcaagctg atcgggaacc cttaatataa 2520

cttcgtataa tgtatgctat acgaagttat taggtccctc gacctgcagc ccaagctagt 2580

gcccgggaat tcgctagggc caccatggac aagcccaaga aaaagcggaa agtgaagtac 2640

agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac 2700

aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg acaggcacag catcaagaag 2760

aacctgatcg gcgccctgct gttcgacagc ggcgaaacag ccgaggccac cagactgaag 2820

agaaccgcca gaagaagata caccaggcgg aagaacagga tctgctatct gcaagagatc 2880

ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc 2940

ctggtggaag aggacaagaa gcacgagaga caccccatct tcggcaacat cgtggacgag 3000

gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc 3060

accgacaagg ccgacctgag actgatctac ctggccctgg cccacatgat caagttcaga 3120

ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc 3180

atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc 3240

gtggacgcca aggctatcct gtctgccaga ctgagcaaga gcagaaggct ggaaaatctg 3300

atcgcccagc tgcccggcga gaagaagaac ggcctgttcg gcaacctgat tgccctgagc 3360

ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag 3420

ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag 3480

tacgccgacc tgttcctggc cgccaagaac ctgtctgacg ccatcctgct gagcgacatc 3540

ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac 3600

gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag 3660

aagtacaaag aaatcttctt cgaccagagc aagaacggct acgccggcta catcgatggc 3720

ggcgctagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc 3780

accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgagaaagca gagaaccttc 3840

3900

3960

ttcaggatcc cctactacgt gggccccctg gccagaggca acagcagatt cgcctggatg 4020

accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc 4080

gccagcgccc agagcttcat cgagagaatg acaaacttcg ataagaacct gcccaacgag 4140

aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtacaa cgagctgacc 4200

aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa 4260

aaggccatcg tggacctgct gttcaagacc aacagaaaag tgaccgtgaa gcagctgaaa 4320

gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat 4380

agattcaacg ccctccctgggg cacataccac gatctgctga aaattatcaa ggacaaggac 4440

ttcctggata acgaagagaa cgaggacatt ctggaagata tcgtgctgac cctgacactg 4500

tttgaggacc gcgagatgat cgaggaaagg ctgaaaacct acgctcacct gttcgacgac 4560

aaagtgatga agcagctgaa gagaaggcgg tacaccggct ggggcaggct gagcagaaag 4620

ctgatcaacg gcatcagaga caagcagagc ggcaagacaa tcctggattt cctgaagtcc 4680

gacggcttcg ccaaccggaa cttcatgcag ctgatccacg acgacagcct gacattcaaa 4740

gaggacatcc agaaagccca ggtgtccggc cagggcgact ctctgcacga gcatatcgct 4800

aacctggccg gcagccccgc tatcaagaag ggcatcctgc agacagtgaa ggtggtggac 4860

gagctcgtga aagtgatggg cagacacaag cccgagaaca tcgtgatcga gatggctaga 4920

gagaaccaga ccacccagaa gggacagaag aactcccgcg agaggatgaa gagaatcgaa 4980

gagggcatca aaagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag 5040

ctgcagaacg agaagctgta cctgtactac ctgcagaatg gccgggatat gtacgtggac 5100

caggaactgg acatcaacag actgtccgac tacgatgtgg accatatcgt gcctcagagc 5160

tttctgaagg acgactccat cgataacaaa gtgctgactc ggagcgacaa gaacagaggc 5220

aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcgacag 5280

ctgctgaacg ccaagctgat tacccagagg aagttcgata acctgaccaa ggccgagaga 5340

ggcggcctga gcgagctgga taaggccggc ttcatcaaga ggcagctggt ggaaaccaga 5400

cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgaa 5460

aacgataagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat 5520

ttcgggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac 5580

gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa 5640

agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc 5700

gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt 5760

ttcaagaccg aaatcaccct ggccaacggc gagatcagaa agcgccctct gatcgagaca 5820

aacggcgaaa ccggggagat cgtgtgggat aagggcagag acttcgccac agtgcgaaag 5880

gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc 5940

agcaaagagt ctatcctgcc caagaggaac agcgacaagc tgatcgccag aaagaaggac 6000

tgggacccca agaagtacgg cggcttcgac agccctaccg tggcctactc tgtgctggtg 6060

gtggctaagg tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg 6120

atcaccatca tggaaagaag cagctttgag aagaacccta tcgactttct ggaagccaag 6180

ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag 6240

ctggaaaacg gcagaaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgag 6300

6360

6420

ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgcc 6480

aatctggaca aggtgctgtc tgcctacaac aagcacaggg acaagcctat cagagagcag 6540

gccgagaata tcatccacct gttcaccctg acaaacctgg gcgctcctgc cgccttcaag 6600

tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc 6660

accctgatcc accagagcat caccggcctg tacgagacaa gaatcgacct gtctcagctg 6720

ggaggcgaca agagacctgc cgccactaag aaggccggac aggccaaaaa gaagaagtga 6780

gtcgacctcg acctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 6840

cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 6900

aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 6960

7020

gcgtgccctt gggctccccg ggcgcggcgg ccatcgctcg agtaaaattg gagggacaag 7080

acttcccaca gattttcggt tttgtcggga agttttttaa taggggcaaa taaggaaaat 7140

gggaggatag gtagtcatct ggggttttat gcagcaaaac tacaggttat tattgcttgt 7200

gatccgc 7207

<210> 18

<211> 4990

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221> misc_feature

<222> (300)..(333)

<223> LoxP

<220>

<221> misc_feature

<222> (382)..(391)

<223> Kozak

<220>

<221> misc_feature

<222> (388)..(4560)

<223> Cas9

<220>

<221> misc_feature

<222> (397)..(417)

<223>Single NLS

<220>

<221> misc_feature

<222> (4513)..(4560)

<223>Double NLS

<220>

<221> misc_feature

<222> (4566)..(4781)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (4841)..(4990)

<223>Mouse downstream Rosa26 sequence

<400> 18

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctagttgaccag 180

240

300

taacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagct 360

420

tacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgag 480

540

aagaacctgatcggcgccctgctgttcgacagcggcgaaacagccgaggccaccagactg 600

660

atcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtcc 720

ttcctggtggaagaggacaagaagcacgagagacaccccatcttcggcaacatcgtggac 780

gaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggac 840

agcaccgacaaggccgacctgagactgatctacctggccctggcccacatgatcaagttc 900

agaggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctg 960

ttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagc 1020

ggcgtggacgccaaggctatcctgtctgccagactgagcaagagcagaaggctggaaaat 1080

1140

agcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactg 1200

1260

cagtacgccgacctgttcctggccgccaagaacctgtctgacgccatcctgctgagcgac 1320

1380

tacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcct 1440

gagaagtacaaagaaatcttcttcgaccagagcaagaacggctacgccggctacatcgat 1500

ggcggcgctagcgggaagagttctacaagttcatcaagcccatcctggaaaagatggac 1560

ggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgagaaagcagagaacc 1620

ttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgctatcctgaga 1680

1740

accttcaggatcccctactacgtgggccccctggccagaggcaacagcagattcgcctgg 1800

1860

ggcgccagcgcccagagcttcatcgagagaatgacaaacttcgataagaacctgcccaac 1920

gagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtacaacgagctg 1980

2040

aaaaaggccatcgtggacctgctgttcaagaccaacagaaaagtgaccgtgaagcagctg 2100

2160

2220

gacttcctggataacgaagagaacgaggacattctggaagatatcgtgctgaccctgaca 2280

2340

gacaaagtgatgaagcagctgaagagaaggcggtacaccggctggggcaggctgagcaga 2400

2460

2520

aaagaggacatccagaaagcccaggtgtccggccagggcgactctctgcacgagcatatc 2580

gctaacctggccggcagccccgctatcaagaagggcatcctgcagacagtgaaggtggtg 2640

2700

agagagaaccagaccacccagaagggacagaagaactcccgcgagaggatgaagagaatc 2760

gaagagggcatcaaagagctgggcagccagatcctgaaagaacacccgtggaaaacacc 2820

cagctgcagaacgagaagctgtacctgtactacctgcagaatggccgggatatgtacgtg 2880

gaccaggaactggacatcaacagactgtccgactacgatgtggaccatatcgtgcctcag 2940

agctttctgaaggacgactccatcgataacaaagtgctgactcggagcgacaagaacaga 3000

ggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagaagatgaagaactactggcga 3060

3120

agaggcggcctgagcgagctggataaggccggcttcatcaagaggcagctggtggaaacc 3180

agacagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgac 3240

gaaaacgataagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtcc 3300

gatttccgggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcc 3360

3420

3480

3540

tttttcaagaccgaaatcaccctggccaacggcgagatcagaaagcgccctctgatcgag 3600

acaaacggcgaaaccggggagatcgtgtgggataagggcagagacttcgccacagtgcga 3660

aaggtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggc 3720

3780

3840

3900

gggatcaccatcatggaaagaagcagctttgagaagaaccctatcgactttctggaagcc 3960

aagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttc 4020

gagctggaaaacggcagaaagagaatgctggcctctgccggcgaactgcagaagggaaac 4080

4140

4200

4260

4320

caggccgagaatatcatccacctgttcaccctgacaaacctgggcgctcctgccgccttc 4380

aagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggac 4440

gccaccctgatccaccagagcatcaccggcctgtacgagacaagaatcgacctgtctcag 4500

4560

tgagtcgacctcgacctcgactgtgccttctagttgccagccatctgttgtttgcccctc 4620

4680

ggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggca 4740

ggacagcaagggggaggattgggaagacaatggcaggcatgctggggaactagtggtgcc 4800

agggcgtgcccttgggctccccgggcgcggcggccatcgctcgagtaaaattggagggac 4860

aagacttcccacagattttcggttttgtcgggaagttttttaataggggcaaataaggaa 4920

aatgggaggataggtagtcatctggggttttatgcagcaaaactacacaggttattattgct 4980

tgtgatccgc 4990

<210> 19

<211> 1391

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 19

Met Asp Lys Pro Lys Lys Lys Arg Lys Val Lys Tyr Ser Ile Gly Leu

1 5 10 15

Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr

20 25 30

Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His

35 40 45

Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu

50 55 60

Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr

65 70 75 80

Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu

85 90 95

Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe

100 105 110

Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn

115 120 125

Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His

130 135 140

Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu

145 150 155 160

Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu

165 170 175

Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe

180 185 190

Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile

195 200 205

Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser

210 215 220

Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys

225 230 235 240

Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr

245 250 255

Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln

260 265 270

Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln

275 280 285

Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser

290 295 300

Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr

305 310 315 320

Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His

325 330 335

Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu

340 345 350

Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly

355 360 365

Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys

370 375 380

Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu

385 390 395 400

Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser

405 410 415

Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg

420 425 430

Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu

435 440 445

Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg

450 455 460

Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile

465 470 475 480

Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln

485 490 495

Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu

500 505 510

Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr

515 520 525

Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro

530 535 540

Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe

545 550 555 560

Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe

565 570 575

Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp

580 585 590

Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile

595 600 605

Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu

610 615 620

Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu

625 630 635 640

Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys

645 650 655

Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys

660 665 670

Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp

675 680 685

Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile

690 695 700

His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val

705 710 715 720

Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly

725 730 735

Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp

740 745 750

Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile

755 760 765

Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser

770 775 780

Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

785 790 795 800

Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu

805 810 815

Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp

820 825 830

Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile

835 840 845

Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu

850 855 860

Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu

865 870 875 880

Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala

885 890 895

Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg

900 905 910

Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu

915 920 925

Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser

930 935 940

Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val

945 950 955 960

Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp

965 970 975

Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His

980 985 990

Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr

995 1000 1005

Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr

1010 1015 1020

Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys

1025 1030 1035

Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe

1040 1045 1050

Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro

1055 1060 1065

Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys

1070 1075 1080

Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln

1085 1090 1095

Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser

1100 1105 1110

Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1115 1120 1125

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1130 1135 1140

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys

1145 1150 1155

Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile

1160 1165 1170

Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe

1175 1180 1185

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile

1190 1195 1200

Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys

1205 1210 1215

Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu

1220 1225 1230

Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His

1235 1240 1245

Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln

1250 1255 1260

Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu

1265 1270 1275

Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn

1280 1285 1290

Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro

1295 1300 1305

Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr

1310 1315 1320

Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile

1325 1330 1335

Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr

1340 1345 1350

Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1355 1360 1365

Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys

1370 1375 1380

Ala Gly Gln Ala Lys Lys Lys Lys

1385 1390

<210> 20

<211> 9673

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221>misc_feature

<222> (205)..(1923)

<223>CAGG promoter

<220>

<221> misc_feature

<222> (2006)..(2039)

<223> LoxP

<220>

<221> misc_feature

<222> (2130)..(4195)

<223> Neo-PolyA

<220>

<221> misc_feature

<222> (4223)..(4256)

<223> LoxP

<220>

<221> misc_feature

<222> (4351)..(4360)

<223> Kozak

<220>

<221> misc_feature

<222> (4360)..(4425)

<223> 3xFLAG

<220>

<221> misc_feature

<222> (4426)..(8598)

<223> Cas9

<220>

<221> misc_feature

<222> (4435)..(4455)

<223>Single NLS

<220>

<221> misc_feature

<222> (8551)..(8598)

<223>Double NLS

<220>

<221> misc_feature

<222> (8645)..(9241)

<223>WPRE

<220>

<221> misc_feature

<222> (9249)..(9464)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (9524)..(9673)

<223>Mouse downstream Rosa26 sequence

<400> 20

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctccttaattaa 180

acgcgtctggcctcgcgagtgtgtactagttattaatagtaatcaattacggggtcatta 240

gttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggc 300

tgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacg 360

ccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttg 420

gcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaa 480

tggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtac 540

atctacgtattagtcatcgctattaccatggtcgaggtgagcccccgttctgcttcact 600

ctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatttt 660

tgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggc 720

gaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctc 780

840

cggcgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcct 900

960

1020

ggctgcgtgaaagccttgaggggctccgggaggggccctttgtgcggggggagcggctcgg 1080

ggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcg 1140

gctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgagggga 1200

1260

1320

1380

1440

cgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggccccc 1500

ggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgt 1560

1620

1680

tgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctc 1740

ggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggct 1800

1860

tcctacagctcctgggcaacgtgctggtttattgtgctgtctcatcattttggcaaagaat 1920

tcgctaggagaattgatttgataccgcgggccctaagtcgacatttaaatcatttaaatc 1980

2040

aggtccctcgacctgcaggaattgttgacaattaatcatcggcatagtatatcggcatag 2100

2160

acgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacaga 2220

2280

ttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctat 2340

2400

gaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttg 2460

ctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatc 2520

cggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga 2580

tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccag 2640

ccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgaccc 2700

atggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcg 2760

actgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgata 2820

ttgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg 2880

ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaggggatc 2940

3000

3060

aaggattggagctacgggggtgggggtggggtgggattagataaatgcctgctctttact 3120

3180

3240

3300

3360

cttcattctcagtattgttttgccaagttctaattccatcagaagcttgcagatctgcga 3420

3480

tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa 3540

ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3600

3660

tcaatgtatc ttatcatgtc tggatctgcg actctagagg atcataatca gccataccac 3720

atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 3780

3840

aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 3900

3960

atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 4020

ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 4080

aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt ttttcactg 4140

cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccccatc 4200

aagctgatcc ggaaccctta atataacttc gtataatgta tgctatacga agttattagg 4260

tccctcgacc tgcagcccaa gctagtgccc gggtaggtcc ctcgacctgc agcccaagct 4320

agatcgaatt cggccggcct tcgaacacgt gccaccatgg actataagga ccacgacgga 4380

4440

aaaaagcgga aagtgaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg 4500

gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc 4560

gacaggcaca gcatcaagaa gaacctgatc ggcgccctgc tgttcgacag cggcgaaaca 4620

gccgaggcca ccagactgaa gagaaccgcc agaagaagat acaccaggcg gaagaacagg 4680

atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc 4740

cacagactgg aagagtcctt cctggtggaa gaggacaaga agcacgagag acaccccatc 4800

ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg 4860

agaaagaaac tggtggacag caccgacaag gccgacctga gactgatcta cctggccctg 4920

gcccacatga tcaagttcag aggccacttc ctgatcgagg gcgacctgaa ccccgacaac 4980

agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa 5040

aaccccatca acgccagcgg cgtggacgcc aaggctatcc tgtctgccag actgagcaag 5100

agcagaaggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa cggcctgttc 5160

ggcaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg 5220

gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg 5280

ctggcccaga tcggcgacca gtacgccgac ctgttcctgg ccgccaagaa cctgtctgac 5340

gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc 5400

gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc 5460

gtgcggcagc agctgcctga gaagtacaaa gaaatcttct tcgaccagag caagaacggc 5520

tacgccggct acatcgatgg cggcgctagc caggaagagt tctacaagtt catcaagccc 5580

atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg 5640

ctgagaaagc agagaacctt cgacaacggc agcatccccc accagatcca cctgggagag 5700

ctgcacgcta tcctgagaag gcaggaagat ttttacccat tcctgaagga caaccgggaa 5760

aagatcgaga agatcctgac cttcaggatc ccctactacg tgggccccct ggccagaggc 5820

aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc 5880

gaggaagtgg tggacaaggg cgccagcgcc cagagcttca tcgagagaat gacaaacttc 5940

gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc 6000

accgtgtaca acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc 6060

ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caacagaaaa 6120

gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg 6180

gaaatctccg gcgtggaaga tagattcaac gcctccctgg gcacatacca cgatctgctg 6240

aaaattatca aggacaagga cttcctggat aacgaagaga acgaggacat tctggaagat 6300

atcgtgctga ccctgacact gtttgaggac cgcgagatga tcgaggaaag gctgaaaacc 6360

tacgctcacc tgttcgacga caaagtgatg aagcagctga agagaaggcg gtacaccggc 6420

tggggcaggc tgagcagaaa gctgatcaac ggcatcagag acaagcagag cggcaagaca 6480

atcctggatt tcctgaagtc cgacggcttc gccaaccgga acttcatgca gctgatccac 6540

6600

tctctgcacg agcatatcgc taacctggcc ggcagccccg ctatcaagaa gggcatcctg 6660

cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gcagacacaa gcccgagaac 6720

atcgtgatcg agatggctag agagaaccag accacccaga agggacagaa gaactcccgc 6780

gagaggatga agagaatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa 6840

caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat 6900

ggccgggata tgtacgtgga cgggaactg gacatcaaca gactgtccga ctacgatgtg 6960

gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgataacaa agtgctgact 7020

cggagcgaca agaacagagg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag 7080

atgaagaact actggcgaca gctgctgaac gccaagctga ttacccagag gaagttcgat 7140

aacctgacca aggccgagag aggcggcctg agcgagctgg ataaggccgg cttcatcaag 7200

aggcagctgg tggaaaccag acagatcaca aagcacgtgg cacagatcct ggactcccgg 7260

atgaacacta agtacgacga aaacgataag ctgatccggg aagtgaaagt gatcaccctg 7320

aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc 7380

aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc 7440

aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg 7500

cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc 7560

tcagcaaca tcatgaactt tttcaagacc gaaatcaccc tggccaacgg cgagatcaga 7620

aagcgccctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggcaga 7680

gacttcgcca cagtgcgaaa ggtgctgagc atgccccaag tgaatatcgt gaaaaagacc 7740

gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgacaag 7800

ctgatcgcca gaaagaagga ctgggacccc aagaagtacg gcggcttcga cagccctacc 7860

gtggcctact ctgtgctggt ggtggctaag gtggaaaagg gcaagtccaa gaaactgaag 7920

agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagctttga gaagaaccct 7980

atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg 8040

cctaagtact ccctgttcga gctggaaaac ggcagaaaga gaatgctggc ctctgccggc 8100

gaactgcaga agggaaacga gctggccctg cctagcaaat atgtgaactt cctgtacctg 8160

8220

gtggaacagc ataagcacta cctggacgag atcatcgagc agatcagcga gttctccaag 8280

agagtgatcc tggccgacgc caatctggac aaggtgctgt ctgcctacaa caagcacagg 8340

gacaagccta tcagagagca ggccgagaat atcatccacc tgttcaccct gacaaacctg 8400

ggcgctcctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc 8460

accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca 8520

agaatcgacc tgtctcagct gggaggcgac aagagacctg ccgccactaa gaaggccgga 8580

caggccaaaa agaagaagtg ataaatgcat ggccggccct gcaggaattc gatatcaagc 8640

ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 8700

atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 8760

cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 8820

aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 8880

cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 8940

ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg 9000

ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt 9060

ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 9120

cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 9180

cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc 9240

gataccgtcg acctcgacct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 9300

ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 9360

tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 9420

gcaggacagc aagggggagg attgggaaga caatggcagg catgctgggg aactagtggt 9480

gccagggcgt gcccttgggc tccccgggcg cggcggccat cgctcgagta aaattggagg 9540

gacaagactt cccacagatt ttcggttttg tcgggaagtt ttttaatagg ggcaaataag 9600

gaaaatggga ggataggtag tcatctgggg ttttatgcag caaaactaca ggttattatt 9660

gcttgtgatccgc 9673

<210> 21

<211> 7456

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221>misc_feature

<222> (1)..(170)

<223>Mouse upstream Rosa26 sequence

<220>

<221> misc_feature

<222> (205)..(1923)

<223>CAGG promoter

<220>

<221> misc_feature

<222> (2006)..(2039)

<223> LoxP

<220>

<221> misc_feature

<222> (2134)..(2143)

<223> Kozak

<220>

<221> misc_feature

<222> (2143)..(2208)

<223> 3xFLAG

<220>

<221> misc_feature

<222> (2209)..(6381)

<223> Cas9

<220>

<221> misc_feature

<222> (2218)..(2238)

<223>Single NLS

<220>

<221> misc_feature

<222> (6334)..(6381)

<223>Double NLS

<220>

<221> misc_feature

<222> (6428)..(7024)

<223>WPRE

<220>

<221> misc_feature

<222> (7032)..(7247)

<223>bGH polyadenylation signal

<220>

<221>misc_feature

<222> (7307)..(7456)

<223>Mouse downstream Rosa26 sequence

<400> 21

ctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgtt 60

gcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctg 120

ggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctccttaattaa 180

acgcgtctggcctcgcgagtgtgtactagttattaatagtaatcaattacggggtcatta 240

gttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggc 300

tgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacg 360

ccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttg 420

gcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaa 480

tggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtac 540

atctacgtattagtcatcgctattaccatggtcgaggtgagcccccgttctgcttcact 600

ctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatttt 660

tgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggc 720

gaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctc 780

840

cggcgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcct 900

960

1020

ggctgcgtgaaagccttgaggggctccgggaggggccctttgtgcggggggagcggctcgg 1080

ggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcg 1140

gctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgagggga 1200

1260

1320

1380

1440

cgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggccccc 1500

ggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgt 1560

1620

1680

tgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctc 1740

ggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggct 1800

1860

tcctacagctcctgggcaacgtgctggtttattgtgctgtctcatcattttggcaaagaat 1920

tcgctaggagaattgatttgataccgcgggccctaagtcgacatttaaatcatttaaatc 1980

2040

2100

2160

ggagactacaaggatcatgatattgattacaaagacgatgacgataagatggacaagccc 2220

aagaaaaagcggaaagtgaagtacagcatcggcctggacatcggcaccaactctgtgggc 2280

2340

accgacaggcacagcatcaagaagaacctgatcggcgccctgctgttcgacagcggcgaa 2400

2460

aggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttc 2520

ttccacagactggaagagtccttcctggtggaagaggacaagaagcacgagagacacccc 2580

atcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccac 2640

ctgagaaagaaactggtggacagcaccgacaaggccgacctgagactgatctacctggcc 2700

2760

aacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgag 2820

gaaaaccccatcaacgccagcggcgtggacgccaaggctatcctgtctgccagactgagc 2880

2940

ttcggcaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgac 3000

3060

ctgctggccgatcggcgaccagtacgccgacctgttcctggccgccaagaacctgtct 3120

3180

agcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagct 3240

3300

3360

cccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggac 3420

ctgctgagaaagcagagaaccttcgacaacggcagcatcccccaccagatccacctggga 3480

gagctgcacgctatcctgagaaggcaggaagatttttacccattcctgaaggacaaccgg 3540

3600

ggcaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaac 3660

3720

3780

ttcaccgtgtacaacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagccc 3840

gccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaacaga 3900

3960

gtggaaatctccggcgtggaagatagattcaacgcctccctgggcacataccacgatctg 4020

4080

gatatcgtgctgaccctgacactgtttgaggaccgcgagatgatcgaggaaaggctgaaa 4140

acctacgctcacctgttcgacgacaaagtgatgaagcagctgaagaagaaggcggtacacc 4200

ggctggggcaggctgagcagaaagctgatcaacggcatcagagacaagcagagcggcaag 4260

acaatcctggatttcctgaagtccgacggcttcgccaaccggaacttcatgcagctgatc 4320

4380

gactctctgcacgagcatatcgctaacctggccggcagccccgctatcaagaagggcatc 4440

4500

aacatcgtgatcgagatggctagagagaaccagaccacccagaagggacagaagaactcc 4560

4620

gaacacccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcag 4680

aatggccgggatatgtacgtggaccaggaactggacatcaacagactgtccgactacgat 4740

gtggaccatatcgtgcctcagagctttctgaaggacgactccatcgataacaaagtgctg 4800

actcggagcgacaagaacagaggcaagagcgacaacgtgccctccgaagaggtcgtgaag 4860

aagatgaagaactactggcgacagctgctgaacgccaagctgattacccagaggaagttc 4920

gataacctgaccaaggccgagagaggcggcctgagcgagctggataaggccggcttcatc 4980

aagaggcagctggtggaaaccagacagatcacaaagcacgtggcacagatcctggactcc 5040

cggatgaacactaagtacgacgaaaacgataagctgatccgggaagtgaaagtgatcacc 5100

5160

5220

5280

5340

ttctacagcaacatcatgaactttttcaagaccgaaatcaccctggccaacggcgagatc 5400

agaaagcgccctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggc 5460

aggacttcgccacagtgcgaaaggtgctgagcatgccccaagtgaatatcgtgaaaaag 5520

accgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgac 5580

aagctgatcgccagaaagaaggactgggaccccaagaagtacggcggcttcgacagccct 5640

accgtggcctactctgtgctggtggtggctaaggtggaaaagggcaagtccaagaaactg 5700

aagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagctttgagaagaac 5760

cctatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaag 5820

ctgcctaagtactccctgttcgagctggaaaacggcagaaagagaatgctggcctctgcc 5880

ggcgaactgcagaagggaaacgagctggccctgcctagcaaatatgtgaacttcctgtac 5940

ctggcctcccactatgagaagctgaagggcagccctgaggacaacgaacagaaacagctg 6000

6060

6120

agggacaagcctatcagagagcaggccgagaatatcatccacctgttcaccctgacaaac 6180

ctgggcgctcctgccgccttcaagtactttgacaccaccatcgaccgggaagaggtacacc 6240

agcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgag 6300

acaagaatcgacctgtctcagctgggaggcgacaagagacctgccgccactaagaaggcc 6360

ggacaggccaaaaagaagaagtgataaatgcatggccggccctgcaggaattcgatatca 6420

agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta 6480

actatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgcta 6540

6600

6660

6720

tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag 6780

gggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttc 6840

cttggctgctcgcctgtgttgccacctggattctgcgcggacgtccttctgctacgtcc 6900

cttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctc 6960

7020

atcgataccgtcgacctcgacctcgactgtgccttctagttgccagccatctgttgtttg 7080

cccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaata 7140

aaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt 7200

ggggcaggacagcaagggggaggattgggaagacaatggcaggcatgctggggaactagt 7260

ggtgccagggcgtgcccttgggctccccgggcgcggcggccatcgctcgagtaaaattgg 7320

agggacaagacttcccacagattttcggttttgtcgggaagtttttttaataggggcaaat 7380

aaggaaaatgggaggataggtagtcatctggggtttttgcagcaaaactacaggttatt 7440

attgcttgtgatccgc 7456

<210> 22

<211> 1413

<212>PRT

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(22)

<223> 3xFLAG

<220>

<221> MISC_FEATURE

<222> (23)..(1413)

<223> Cas9

<400> 22

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys Met Asp Lys Pro Lys Lys Lys Arg Lys Val

20 25 30

Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala

35 40 45

Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu

50 55 60

Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu

65 70 75 80

Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr

85 90 95

Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln

100 105 110

Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His

115 120 125

Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg

130 135 140

His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys

145 150 155 160

Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp

165 170 175

Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys

180 185 190

Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser

195 200 205

Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu

210 215 220

Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile

225 230 235 240

Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala

245 250 255

Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala

260 265 270

Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala

275 280 285

Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu

290 295 300

Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu

305 310 315 320

Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg

325 330 335

Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys

340 345 350

Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val

355 360 365

Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser

370 375 380

Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu

385 390 395 400

Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu

405 410 415

Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg

420 425 430

Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu

435 440 445

His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp

450 455 460

Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr

465 470 475 480

Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg

485 490 495

Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp

500 505 510

Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp

515 520 525

Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr

530 535 540

Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr

545 550 555 560

Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala

565 570 575

Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln

580 585 590

Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu

595 600 605

Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His

610 615 620

Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu

625 630 635 640

Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu

645 650 655

Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe

660 665 670

Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp

675 680 685

Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser

690 695 700

Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg

705 710 715 720

Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp

725 730 735

Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His

740 745 750

Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln

755 760 765

Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys

770 775 780

Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln

785 790 795 800

Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly

805 810 815

Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn

820 825 830

Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly

835 840 845

Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp

850 855 860

Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser

865 870 875 880

Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser

885 890 895

Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

900 905 910

Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn

915 920 925

Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly

930 935 940

Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val

945 950 955 960

Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp

965 970 975

Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val

980 985 990

Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn

995 1000 1005

Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly

1010 1015 1020

Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

1025 1030 1035

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1040 1045 1050

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr

1055 1060 1065

Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn

1070 1075 1080

Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1085 1090 1095

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg

1100 1105 1110

Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu

1115 1120 1125

Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg

1130 1135 1140

Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys

1145 1150 1155

Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu

1160 1165 1170

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser

1175 1180 1185

Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe

1190 1195 1200

Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu

1205 1210 1215

Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe

1220 1225 1230

Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu

1235 1240 1245

Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn

1250 1255 1260

Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1265 1270 1275

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1280 1285 1290

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg

1295 1300 1305

Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr

1310 1315 1320

Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1325 1330 1335

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe

1340 1345 1350

Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr

1355 1360 1365

Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly

1370 1375 1380

Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys

1385 1390 1395

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1400 1405 1410

<210> 23

<211> 22

<212> PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 23

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys

twenty

<210> 24

<211> 238

<212> PRT

<213>Artificial sequence

<220>

<223>Synthetic

<400> 24

Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

20 25 30

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

65 70 75 80

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

85 90 95

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 25

<211> 16

<212>RNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 25

guuuuuagagcuaugcu 16

<210> 26

<211> 67

<212>RNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 26

agcauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucg 60

67

<210> 27

<211> 77

<212>RNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 27

60

ggcaccgagucggugcu 77

<210> 28

<211> 4953

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> misc_feature

<222> (1)..(4173)

<223> Cas9

<220>

<221> misc_feature

<222> (4174)..(4239)

<223> P2A

<220>

<221> misc_feature

<222> (4240)..(4953)

<223> eGFP

<400> 28

atggacaagc ccaagaaaaa gcggaaagtg aagtacagca tcggcctgga catcggcacc 60

aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag 120

gtgctgggca acaccgacag gcacagcatc aagaagaacc tgatcggcgc cctgctgttc 180

gacagcggcg aaacagccga ggccaccaga ctgaagagaa ccgccagaag aagatacacc 240

aggcggaaga acaggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg 300

gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga caagaagcac 360

gagagacacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 420

accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgagactg 480

atctacctgg ccctggccca catgatcaag ttcagaggcc acttcctgat cgagggcgac 540

ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 600

cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc tatcctgtct 660

gccagactga gcaagagcag aaggctggaa aatctgatcg cccagctgcc cggcgagaag 720

aagaacggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag 780

agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 840

gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt cctggccgcc 900

aagaacctgt ctgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc 960

aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc 1020

ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagaaat cttcttcgac 1080

cagagcaaga acggctacgc cggctacatc gatggcggcg ctagccagga agagttctac 1140

aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg 1200

aacagagagg acctgctgag aaagcagaga accttcgaca acggcagcat cccccaccag 1260

atccacctgg gagagctgca cgctatcctg agaaggcagg aagattttta cccattcctg 1320

aaggacaacc gggaaaagat cgagaagatc ctgaccttca ggatccccta ctacgtggggc 1380

cccctggcca gaggcaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc 1440

accccctgga acttcgagga agtggtggac aagggcgcca gcgcccagag cttcatcgag 1500

agaatgacaa acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 1560

ctgtacgagt acttcaccgt gtacaacgag ctgaccaaag tgaaatacgt gaccgaggga 1620

atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc 1680

aagaccaaca gaaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag 1740

tgcttcgact ccgtggaaat ctccggcgtg gaagatagat tcaacgcctc cctgggcaca 1800

taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggataacga agagaacgag 1860

gacattctgg aagatatcgt gctgaccctg acactgtttg aggaccgcga gatgatcgag 1920

gaaaggctga aaacctacgc tcacctgttc gacgacaaag tgatgaagca gctgaagaga 1980

aggcggtaca ccggctgggg caggctgagc agaaagctga tcaacggcat cagagacaag 2040

cagagcggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa ccggaacttc 2100

atgcagctga tccacgacga cagcctgaca ttcaaagagg acatccagaa agcccaggtg 2160

tccggccagg gcgactctct gcacgagcat atcgctaacc tggccggcag ccccgctatc 2220

aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggcaga 2280

cacaagcccg agaacatcgt gatcgagatg gctagagaga accagaccac ccagaaggga 2340

cagaagaact cccgcgagag gatgaagaga atcgaagagg gcatcaaaga gctgggcagc 2400

cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg 2460

tactacctgc agaatggccg ggatatgtac gtggaccagg aactggacat caacagactg 2520

2580

aacaaagtgc tgactcggag cgacaagaac agaggcaaga gcgacaacgt gccctccgaa 2640

gaggtcgtga agaagatgaa gaactactgg cgacagctgc tgaacgccaa gctgattacc 2700

cagaggaagt tcgataacct gaccaaggcc gagagaggcg gcctgagcga gctggataag 2760

gccggcttca tcaagaggca gctggtggaa accagacaga tcacaaagca cgtggcacag 2820

atcctggact cccggatgaa cactaagtac gacgaaaacg ataagctgat ccgggaagtg 2880

aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac 2940

aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg 3000

3060

aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc 3120

gccaagtact tcttctacag caacatcatg aactttttca agaccgaaat caccctggcc 3180

aacggcgaga tcagaaagcg ccctctgatc gagacaaacg gcgaaaccgg ggagatcgtg 3240

tgggataagg gcagagactt cgccacagtg cgaaaggtgc tgagcatgcc ccaagtgaat 3300

atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag 3360

aggaacagcg acaagctgat cgccagaaag aaggactggg accccaagaa gtacggcggc 3420

ttcgacagcc ctaccgtggc ctactctgtg ctggtggtgg ctaaggtgga aaagggcaag 3480

tccaagaaac tgaaagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc 3540

tttgagaaga accctatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac 3600

ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggcag aaagagaatg 3660

ctggcctctg ccggcgaact gcagaaggga aacgagctgg ccctgcctag caaatatgtg 3720

aacttcctgt acctggcctc ccactatgag aagctgaagg gcagccctga ggacaacgaa 3780

cagaaacagc tgtttgtgga acagcataag cactacctgg acgagatcat cgagcagatc 3840

agcgagttct ccaagagagt gatcctggcc gacgccaatc tggacaaggt gctgtctgcc 3900

tacaacaagc acagggacaa gcctatcaga gagcaggccg agaatatcat ccacctgttc 3960

accctgacaa acctgggcgc tcctgccgcc ttcaagtact ttgacaccac catcgaccgg 4020

aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc 4080

ggcctgtacg agacaagaat cgacctgtct cagctgggag gcgacaagag acctgccgcc 4140

actaagaagg ccggacaggc caaaaagaag aagggaagcg gagccactaa cttctccctg 4200

ttgaaacaag caggggatgt cgaagagaat cccgggccag tgagcaaggg cgaggagctg 4260

ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 4320

agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 4380

tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 4440

gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 4500

atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 4560

acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 4620

atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 4680

cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 4740

cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 4800

atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 4860

agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 4920

gggatcactc tcggcatgga cgagctgtac aag 4953

<210> 29

<211> 5019

<212> DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> misc_feature

<222> (1)..(66)

<223> 3xFLAG

<220>

<221> misc_feature

<222> (67)..(4239)

<223> Cas9

<220>

<221> misc_feature

<222> (4240)..(4305)

<223> P2A

<220>

<221> misc_feature

<222> (4306)..(5019)

<223> eGFP

<400> 29

gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac 60

gataagatgg acaagcccaa gaaaaagcgg aaagtgaagt acagcatcgg cctggacatc 120

ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa 180

ttcaaggtgc tgggcaacac cgacaggcac agcatcaaga agaacctgat cggcgccctg 240

ctgttcgaca gcggcgaaac agccgaggcc accagactga agagaaccgc cagaagaaga 300

tacaccaggc ggaagaacag gatctgctat ctgcaagaga tcttcagcaa cgagatggcc 360

aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggacaag 420

aagcacgaga gacaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag 480

taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg 540

agactgatct acctggccct ggcccacatg atcaagttca gaggccactt cctgatcgag 600

ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc 660

tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggctatc 720

ctgtctgcca gactgagcaa gagcagaagg ctggaaaatc tgatcgccca gctgcccggc 780

gagaagaaga acggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac 840

ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac 900

gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgttcctg 960

gccgccaaga acctgtctga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag 1020

atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac 1080

ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agaaatcttc 1140

ttcgaccaga gcaagaacgg ctacgccggc tacatcgatg gcggcgctag cggaagag 1200

ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 1260

aagctgaaca gagaggacct gctgagaaag cagagaacct tcgacaacgg cagcatcccc 1320

caccagatcc acctgggaga gctgcacgct atcctgagaa ggcaggaaga tttttaccca 1380

ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttcaggat cccctactac 1440

gtgggccccc tggccagagg caacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 1500

accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc 1560

atcgagagaa tgacaaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 1620

agcctgctgt acgagtactt caccgtgtac aacgagctga ccaaagtgaa atacgtgacc 1680

gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg 1740

ctgttcaaga ccaacagaaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 1800

atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atagattcaa cgcctccctg 1860

ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga taacgaagag 1920

aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga ccgcgagatg 1980

atcgaggaaa ggctgaaaac ctacgctcac ctgttcgacg acaaagtgat gaagcagctg 2040

aagagaaggc ggtacaccgg ctggggcagg ctgagcagaa agctgatcaa cggcatcaga 2100

gacaagcaga gcggcaagac aatcctggat ttcctgaagt cgacggctt cgccaaccgg 2160

aacttcatgc agctgatcca cgacgacagc ctgacattca aagggacat ccagaaagcc 2220

caggtgtccg gccagggcga ctctctgcac gagcatatcg ctaacctggc cggcagcccc 2280

gctatcaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 2340

ggcagacaca agcccgagaa catcgtgatc gagatggcta gagagaacca gaccacccag 2400

2460

ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 2520

tacctgtact acctgcagaa tggccgggat atgtacgtgg accaggaact ggacatcaac 2580

agactgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc 2640

atcgataaca aagtgctgac tcggagcgac aagaacagag gcaagagcga caacgtgccc 2700

tccgaagagg tcgtgaagaa gatgaagaac tactggcgac agctgctgaa cgccaagctg 2760

attacccaga ggaagttcga taacctgacc aaggccgaga gaggcggcct gagcgagctg 2820

gataaggccg gcttcatcaa gaggcagctg gtggaaacca gacagatcac aaagcacgtg 2880

gcacagatcc tggactcccg gatgaacact aagtacgacg aaaacgataa gctgatccgg 2940

gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 3000

3060

gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc 3120

3180

gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgaaatcacc 3240

ctggccaacg gcgagatcag aaagcgccct ctgatcgaga caaacggcga aaccggggag 3300

atcgtgtggg ataagggcag agacttcgcc acagtgcgaa aggtgctgag catgccccaa 3360

gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 3420

cccaagagga acagcgacaa gctgatcgcc agaaagaagg actgggaccc caagaagtac 3480

ggcggcttcg acagccctac cgtggcctac tctgtgctgg tggtggctaa ggtggaaaag 3540

ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga 3600

agcagctttg agaagaaccc tatcgacttt ctggaagcca agggctacaa agaagtgaaa 3660

aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggcagaaag 3720

agaatgctgg cctctgccgg cgaactgcag aagggaaacg agctggccct gcctagcaaa 3780

tatgtgaact tcctgtacct ggcctcccac tatgagaagc tgaagggcag ccctgaggac 3840

aacgaacaga aacagctgtt tgtggaacag cataagcact acctggacga gatcatcgag 3900

cagatcagcg agttctccaa gagagtgatc ctggccgacg ccaatctgga caaggtgctg 3960

tctgcctaca acaagcacag ggacaagcct atcagagagc aggccgagaa tatcatccac 4020

ctgttcaccc tgacaaacct gggcgctcct gccgccttca agtactttga caccaccatc 4080

gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc 4140

atcaccggcc tgtacgagac aagaatcgac ctgtctcagc tgggaggcga caagagacct 4200

gccgccacta agaaggccgg acaggccaaa aagaagaagg gaagcggagc cactaacttc 4260

tccctgttga aacaagcagg ggatgtcgaa gagaatcccg ggccagtgag caagggcgag 4320

gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 4380

aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 4440

ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 4500

tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 4560

tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 4620

tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 4680

aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 4740

aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc 4800

4860

acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 4920

gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 4980

gccgccggga tcactctcgg catggacgag ctgtacaag 5019

<210> 30

<211> 4173

<212> DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 30

atggacaagc ccaagaaaaa gcggaaagtg aagtacagca tcggcctgga catcggcacc 60

aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag 120

gtgctgggca acaccgacag gcacagcatc aagaagaacc tgatcggcgc cctgctgttc 180

gacagcggcg aaacagccga ggccaccaga ctgaagagaa ccgccagaag aagatacacc 240

aggcggaaga acaggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg 300

gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga caagaagcac 360

gagagacacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 420

accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgagactg 480

atctacctgg ccctggccca catgatcaag ttcagaggcc acttcctgat cgagggcgac 540

ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 600

cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc tatcctgtct 660

gccagactga gcaagagcag aaggctggaa aatctgatcg cccagctgcc cggcgagaag 720

aagaacggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag 780

agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 840

gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt cctggccgcc 900

aagaacctgt ctgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc 960

aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc 1020

ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagaaat cttcttcgac 1080

cagagcaaga acggctacgc cggctacatc gatggcggcg ctagccagga agagttctac 1140

aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg 1200

aacagagagg acctgctgag aaagcagaga accttcgaca acggcagcat cccccaccag 1260

atccacctgg gagagctgca cgctatcctg agaaggcagg aagattttta cccattcctg 1320

aaggacaacc gggaaaagat cgagaagatc ctgaccttca ggatccccta ctacgtggggc 1380

cccctggcca gaggcaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc 1440

accccctgga acttcgagga agtggtggac aagggcgcca gcgcccagag cttcatcgag 1500

agaatgacaa acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 1560

ctgtacgagt acttcaccgt gtacaacgag ctgaccaaag tgaaatacgt gaccgaggga 1620

atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc 1680

aagaccaaca gaaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag 1740

tgcttcgact ccgtggaaat ctccggcgtg gaagatagat tcaacgcctc cctgggcaca 1800

taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggataacga agagaacgag 1860

gacattctgg aagatatcgt gctgaccctg acactgtttg aggaccgcga gatgatcgag 1920

gaaaggctga aaacctacgc tcacctgttc gacgacaaag tgatgaagca gctgaagaga 1980

aggcggtaca ccggctgggg caggctgagc agaaagctga tcaacggcat cagagacaag 2040

cagagcggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa ccggaacttc 2100

atgcagctga tccacgacga cagcctgaca ttcaaagagg acatccagaa agcccaggtg 2160

tccggccagg gcgactctct gcacgagcat atcgctaacc tggccggcag ccccgctatc 2220

aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggcaga 2280

cacaagcccg agaacatcgt gatcgagatg gctagagaga accagaccac ccagaaggga 2340

cagaagaact cccgcgagag gatgaagaga atcgaagagg gcatcaaaga gctgggcagc 2400

cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg 2460

tactacctgc agaatggccg ggatatgtac gtggaccagg aactggacat caacagactg 2520

2580

aacaaagtgc tgactcggag cgacaagaac agaggcaaga gcgacaacgt gccctccgaa 2640

gaggtcgtga agaagatgaa gaactactgg cgacagctgc tgaacgccaa gctgattacc 2700

cagaggaagt tcgataacct gaccaaggcc gagagaggcg gcctgagcga gctggataag 2760

gccggcttca tcaagaggca gctggtggaa accagacaga tcacaaagca cgtggcacag 2820

atcctggact cccggatgaa cactaagtac gacgaaaacg ataagctgat ccgggaagtg 2880

aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac 2940

aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg 3000

3060

aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc 3120

gccaagtact tcttctacag caacatcatg aactttttca agaccgaaat caccctggcc 3180

aacggcgaga tcagaaagcg ccctctgatc gagacaaacg gcgaaaccgg ggagatcgtg 3240

tgggataagg gcagagactt cgccacagtg cgaaaggtgc tgagcatgcc ccaagtgaat 3300

atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag 3360

aggaacagcg acaagctgat cgccagaaag aaggactggg accccaagaa gtacggcggc 3420

ttcgacagcc ctaccgtggc ctactctgtg ctggtggtgg ctaaggtgga aaagggcaag 3480

tccaagaaac tgaaagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc 3540

tttgagaaga accctatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac 3600

ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggcag aaagagaatg 3660

ctggcctctg ccggcgaact gcagaaggga aacgagctgg ccctgcctag caaatatgtg 3720

aacttcctgt acctggcctc ccactatgag aagctgaagg gcagccctga ggacaacgaa 3780

cagaaacagc tgtttgtgga acagcataag cactacctgg acgagatcat cgagcagatc 3840

agcgagttct ccaagagagt gatcctggcc gacgccaatc tggacaaggt gctgtctgcc 3900

tacaacaagc acagggacaa gcctatcaga gagcaggccg agaatatcat ccacctgttc 3960

accctgacaa acctgggcgc tcctgccgcc ttcaagtact ttgacaccac catcgaccgg 4020

aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc 4080

ggcctgtacg agacaagaat cgacctgtct cagctgggag gcgacaagag acctgccgcc 4140

actaagaagg ccggacaggc caaaaagaag aag 4173

<210> 31

<211> 4239

<212> DNA

<213>Artificial sequence

<220>

<223>Synthetic

<220>

<221> misc_feature

<222> (1)..(66)

<223> 3xFLAG

<220>

<221> misc_feature

<222> (67)..(4239)

<223> Cas9

<400> 31

gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac 60

gataagatgg acaagcccaa gaaaaagcgg aaagtgaagt acagcatcgg cctggacatc 120

ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa 180

ttcaaggtgc tgggcaacac cgacaggcac agcatcaaga agaacctgat cggcgccctg 240

ctgttcgaca gcggcgaaac agccgaggcc accagactga agagaaccgc cagaagaaga 300

tacaccaggc ggaagaacag gatctgctat ctgcaagaga tcttcagcaa cgagatggcc 360

aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggacaag 420

aagcacgaga gacaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag 480

taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg 540

agactgatct acctggccct ggcccacatg atcaagttca gaggccactt cctgatcgag 600

ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc 660

tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggctatc 720

ctgtctgcca gactgagcaa gagcagaagg ctggaaaatc tgatcgccca gctgcccggc 780

gagaagaaga acggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac 840

ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac 900

gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgttcctg 960

gccgccaaga acctgtctga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag 1020

atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac 1080

ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agaaatcttc 1140

ttcgaccaga gcaagaacgg ctacgccggc tacatcgatg gcggcgctag cggaagag 1200

ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 1260

aagctgaaca gagaggacct gctgagaaag cagagaacct tcgacaacgg cagcatcccc 1320

caccagatcc acctgggaga gctgcacgct atcctgagaa ggcaggaaga tttttaccca 1380

ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttcaggat cccctactac 1440

gtgggccccc tggccagagg caacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 1500

accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc 1560

atcgagagaa tgacaaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 1620

agcctgctgt acgagtactt caccgtgtac aacgagctga ccaaagtgaa atacgtgacc 1680

gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg 1740

ctgttcaaga ccaacagaaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 1800

atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atagattcaa cgcctccctg 1860

ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga taacgaagag 1920

aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga ccgcgagatg 1980

atcgaggaaa ggctgaaaac ctacgctcac ctgttcgacg acaaagtgat gaagcagctg 2040

aagagaaggc ggtacaccgg ctggggcagg ctgagcagaa agctgatcaa cggcatcaga 2100

gacaagcaga gcggcaagac aatcctggat ttcctgaagt cgacggctt cgccaaccgg 2160

aacttcatgc agctgatcca cgacgacagc ctgacattca aagggacat ccagaaagcc 2220

caggtgtccg gccagggcga ctctctgcac gagcatatcg ctaacctggc cggcagcccc 2280

gctatcaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 2340

ggcagacaca agcccgagaa catcgtgatc gagatggcta gagagaacca gaccacccag 2400

2460

ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 2520

tacctgtact acctgcagaa tggccgggat atgtacgtgg accaggaact ggacatcaac 2580

agactgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc 2640

atcgataaca aagtgctgac tcggagcgac aagaacagag gcaagagcga caacgtgccc 2700

tccgaagagg tcgtgaagaa gatgaagaac tactggcgac agctgctgaa cgccaagctg 2760

attacccaga ggaagttcga taacctgacc aaggccgaga gaggcggcct gagcgagctg 2820

gataaggccg gcttcatcaa gaggcagctg gtggaaacca gacagatcac aaagcacgtg 2880

gcacagatcc tggactcccg gatgaacact aagtacgacg aaaacgataa gctgatccgg 2940

gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 3000

3060

gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc 3120

3180

gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgaaatcacc 3240

ctggccaacg gcgagatcag aaagcgccct ctgatcgaga caaacggcga aaccggggag 3300

atcgtgtggg ataagggcag agacttcgcc acagtgcgaa aggtgctgag catgccccaa 3360

gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 3420

cccaagagga acagcgacaa gctgatcgcc agaaagaagg actgggaccc caagaagtac 3480

ggcggcttcg acagccctac cgtggcctac tctgtgctgg tggtggctaa ggtggaaaag 3540

ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga 3600

agcagctttg agaagaaccc tatcgacttt ctggaagcca agggctacaa agaagtgaaa 3660

aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggcagaaag 3720

agaatgctgg cctctgccgg cgaactgcag aagggaaacg agctggccct gcctagcaaa 3780

tatgtgaact tcctgtacct ggcctcccac tatgagaagc tgaagggcag ccctgaggac 3840

aacgaacaga aacagctgtt tgtggaacag cataagcact acctggacga gatcatcgag 3900

cagatcagcg agttctccaa gagagtgatc ctggccgacg ccaatctgga caaggtgctg 3960

tctgcctaca acaagcacag ggacaagcct atcagagagc aggccgagaa tatcatccac 4020

ctgttcaccc tgacaaacct gggcgctcct gccgccttca agtactttga caccaccatc 4080

gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc 4140

atcaccggcc tgtacgagac aagaatcgac ctgtctcagc tgggaggcga caagagacct 4200

gccgcacta agaaggccgg acaggccaaa aagaagaag 4239

<210> 32

<211> 66

<212> DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 32

ggaagcggag ccactaactt ctccctgttg aaacaagcag gggatgtcga agagaatccc 60

gggcca 66

<210> 33

<211> 714

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 33

gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggc 60

120

aagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctc 180

gtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcag 240

cacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttc 300

360

aaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaag 420

ctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggc 480

atcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgac 540

cactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactac 600

ctgagcaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctg 660

ctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag 714

<210> 34

<211> 66

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 34

gactataaggacccgacggagactacaaggatcatgatattgattacaaagacgatgac 60

gataag 66

<210> 35

<211> 597

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 35

cgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgt 60

tgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttc 120

ccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgagga 180

240

cactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccct 300

360

gctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggct 420

gctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggc 480

cctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcg 540

tcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcg 597

<210> 36

<211> 216

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 36

cgacctcgacctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccg 60

120

ttgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggaca 180

gcaagggggaggattgggaagacaatggcaggcatg 216

<210> 37

<211> 2066

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 37

atgggatcggccattgaacaagatggattgcacgcaggttctccggccgcttgggtggag 60

aggctattcggctatgactggggcacaacagacaatcggctgctctgatgccgccgtgttc 120

180

aatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgc 240

gcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtg 300

ccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggct 360

gatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcg 420

480

ctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgc 540

atgccgacggcgatgatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatg 600

660

tatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggct 720

780

cgccttcttgacgagttcttctgaggggatccgctgtaagtctgcagaaattgatgatct 840

attaaacaataaagatgtccactaaaatggaagttttttcctgtcatactttgttaagaag 900

ggtgagaacagagtacctacattttgaatggaaggattggagctacgggggtgggggtgg 960

ggtgggattagataaatgcctgctctttactgaaggctctttactattgctttatgataa 1020

1080

cctcccactcatgatctatagatctatagatctctcgtgggatcattgttttctcttga 1140

ttcccactttgtggttctaagtactgtggtttccaaatgtgtcagtttcatagcctgaag 1200

aacgagatcagcagcctctgttccacatacacttcattctcagtattgttttgccaagtt 1260

ctaattccatcagaagcttgcagatctgcgactctgaggatctgcgactctagaggatc 1320

ataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctc 1380

cccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagct 1440

tataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttca 1500

1560

1620

acctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaact 1680

tgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaata 1740

aagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatc 1800

atgtctggat ctgcgactct agaggatcat aatcagccat accacatttg tagaggtttt 1860

acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat 1920

tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 1980

aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 2040

caatgtatcttatcatgtctggatcc 2066

<210> 38

<211> 1719

<212>DNA

<213>Artificial sequence

<220>

<223>Synthetic

<400> 38

actagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttc 60

cgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgccca 120

ttgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgt 180

240

ccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccag 300

tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 360

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 420

cccccaattt tgtatttatt tattttttaa ttatttttgtg cagcgatgggg ggcggggggg 480

gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 540

agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 600

660

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 720

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 780

agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc 840

tccgggggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg 900

tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg 960

cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc 1020

ggtgcgggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt 1080

1140

ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg 1200

ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg 1260

ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg 1320

1380

tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc 1440

gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1500

cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct 1560

gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 1620

gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc 1680

tggttattgt gctgtctcat cattttggca aagaattcg 1719

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Claims (99)

1. A method for testing the ability of a CRISPR/Cas9 nuclease containing the Cas9 protein and guide RNA to modify a target genomic locus in vivo, comprising: (a) administering to the mouse a guide RNA designed to target the target sequence for the guide RNA at the target genomic locus, wherein the mouse contains in its germline a genomically integrated Cas9 expression cassette at the Rosa26 locus, wherein the Cas9 expression cassette contains the coding sequence for the Cas9 protein, wherein the Cas9 protein is expressed and contains the complete sequence shown in SEQ ID NO: 19, and wherein the guide RNA is administered via adeno-associated virus (AAV) mediated delivery; and (b) assessing the modification of the target genomic locus using CRISPR/Cas9 nuclease. 2. The method of claim 1, wherein the AAV is AAV7, AAV8, or AAV9 and step (b) involves assessing the modification of a target genomic locus in the liver. 3. The method of claim 2 wherein the AAV is AAV8. 4. The method according to any one of the preceding claims, wherein the route of administration of the mouse AAV is intravenous injection, intraparenchymal injection, intraperitoneal injection, nasal instillation, or intravitreal injection. 5. The method according to any one of the preceding claims, wherein the exogenous donor nucleic acid is introduced in step (a), wherein the exogenous donor nucleic acid is intended to recombine with the target genomic locus. 6. The method of claim 5 wherein the exogenous donor nucleic acid is a single stranded oligodeoxynucleotide (ssODN). 7. The method according to any one of the preceding claims, wherein the target genomic locus contains the target gene, and step (b) comprises measuring the expression of the target gene or the activity of a protein encoded by the target gene. 8. The method according to any one of the preceding claims, wherein step (b) comprises sequencing the target genomic locus in one or more cells isolated from a mouse. 9. A method according to any one of the preceding claims, wherein step (b) comprises isolating a target organ or tissue from a mouse and assessing the modification of a target genomic locus in the target organ or tissue. 10. The method of claim 9, wherein step (b) involves assessing the modification of the target genomic locus in two or more different cell types within the target organ or tissue. 11. The method of any one of the preceding claims, wherein step (b) comprises isolating a non-target organ or tissue from a mouse and assessing the modification of a target genomic locus in the non-target organ or tissue. 12. The method of any one of the preceding claims, wherein the Cas9 expression cassette further comprises a polyadenylation signal upstream of the coding sequence for the Cas9 protein, wherein the polyadenylation signal is flanked by recombinase recognition sites, and wherein the polyadenylation signal in the Cas9 expression cassette has been excised in a tissue-specific manner. 13. The method of claim 12, wherein the polyadenylation signal upstream of the coding sequence for the Cas9 protein has been excised in the liver. 14. The method of claim 12 or 13, wherein the recombinase that recognizes the recombinase recognition sites in the Cas9 expression cassette is a Cre recombinase. 15. The method of claim 14, wherein the mouse further comprises a genomically integrated Cre recombinase expression cassette comprising a Cre recombinase coding sequence operably linked to a tissue-specific promoter. 16. The method of claim 14 or 15, wherein the Cre recombinase gene is operably linked to an albumin promoter. 17. The method according to any one of paragraphs. 1-11, wherein the Cas9 expression cassette further comprises a polyadenylation signal upstream of the coding sequence for the Cas9 protein, wherein the polyadenylation signal is flanked by recombinase recognition sites, and wherein the method further comprises administering the mouse recombinase in a tissue-specific manner. 18. The method of claim 17, wherein the recombinase is administered via adeno-associated virus (AAV) mediated delivery or lipid nanoparticle (LNP) mediated delivery. 19. A method according to any one of the preceding claims, wherein the Cas9 expression cassette further comprises a fluorescent protein coding sequence. 20. The method of claim 19, wherein the Cas9 expression cassette comprises a multicistronic nucleic acid comprising a coding sequence for the Cas9 protein and a fluorescent protein coding sequence separated by an internal ribosome entry intermediate site (IRES) or a 2A peptide coding intermediate. 21. The method of claim 20, wherein the multicistronic nucleic acid in the Cas9 expression cassette comprises a coding sequence for the Cas9 protein and a green fluorescent protein coding sequence separated by an intermediate P2A peptide coding sequence. 22. The method according to any one of paragraphs. 1-18, in which the Cas9 expression cassette additionally does not contain a fluorescent protein coding sequence. 23. The method of any one of the preceding claims, wherein the Cas9 protein contains a protein tag. 24. The method of any one of the preceding claims, wherein the 5' end of the Cas9 expression cassette further comprises a 3' splicing sequence. 25. A method according to any one of the preceding claims, wherein the Cas9 expression cassette is operably linked to an endogenous promoter. 26. The method according to any one of paragraphs. 1-24, in which the Cas9 expression cassette is operably linked to an exogenous, constitutive promoter. 27. The method of any one of the preceding claims, wherein the Cas9 expression cassette encodes a protein comprising the sequence shown in SEQ ID NO: 13, 16, or 22. 28. The method of claim 27, wherein the Cas9 expression cassette contains the sequence shown in SEQ ID NO: 28, 29, 30, or 31. 29. The method of claim 28, wherein the Cas9 expression cassette contains the sequence shown in SEQ ID NOs: 1, 12, 14, 15, 17, 18, 20, or 21. 30. A method according to any one of the preceding claims, wherein the Cas9 expression cassette is integrated into the first intron of the Rosa26 locus. 31. The method of any one of the preceding claims, wherein the mouse is heterozygous for the Cas9 expression cassette. 32. The method according to any one of paragraphs. 1-30 in which the mouse is homozygous for the Cas9 expression cassette. 33. The method according to any of the previous paragraphs, wherein the AAV is AAV8 delivered to the mouse by intravenous injection, wherein the Cas9 expression cassette is operably linked to the endogenous Rosa26 promoter, is inserted into the first intron of the Rosa26 locus, and contains, from 5' to 3', the following: (i) 3' splicing sequence; and (ii) a coding sequence for the Cas9 protein, and and stage (b) involves assessing the modification of the target genomic locus in the mouse liver. 34. A method for optimizing the ability of CRISPR/Cas9 nuclease to modify a target genomic locus in vivo, comprising: (I) carrying out the method according to any of the previous paragraphs for the first time in the first mouse; (II) changing the variable and running the method of step (I) a second time with the changed variable in a second mouse; and: (A) the modified variable is: (i) AAV serotype; (ii) the route of administration of the guide RNA administered to the mouse; (iii) the concentration or amount of guide RNA administered to the mouse; or (iv) a guide RNA sequence administered to a mouse, or (B) step (a) further involves administering an exogenous donor nucleic acid, and the altered variable is: (i) a delivery method or route of administration for introducing the exogenous donor nucleic acid into the mouse; (ii) the concentration or amount of exogenous donor nucleic acid administered to the mouse; (iii) the concentration or amount of guide RNA administered to the mouse relative to the concentration or amount of exogenous donor nucleic acid administered to the mouse; or (iv) an exogenous donor nucleic acid sequence introduced into the mouse; and (III) comparing the modification of the target genomic locus in step (I) with the modification of the target genomic locus in step (II), and selecting a method resulting in a modification of the target genomic locus having one or more of the following: increased efficiency, increased accuracy, increased consistency or increased specificity. 35. The method of claim 34 wherein the altered variable in step (II) is an AAV serotype. 36. The method of claim 34, wherein the altered variable in step (II) is the route of administration of the guide RNA administered to the mouse. 37. The method of claim 34, wherein the variable in step (II) is the concentration or amount of guide RNA administered to the mouse. 38. The method of claim 34 wherein the altered variable in step (II) is a guide RNA administered to the mouse. 39. The method of claim 34, wherein the method comprises administering an exogenous donor nucleic acid, and wherein the altered variable in step (II) is a delivery method for administering the exogenous mouse donor nucleic acid. 40. The method of claim 34, wherein the method comprises administering an exogenous donor nucleic acid and the altered variable in step (II) is the route of administration of the exogenous donor nucleic acid administered to the mouse. 41. The method of claim 34, wherein the method comprises administering an exogenous donor nucleic acid, and the altered variable in step (II) is the concentration or amount of exogenous donor nucleic acid administered to the mouse. 42. The method of claim 34, wherein the method comprises administering an exogenous donor nucleic acid, and the altered variable in step (II) is the concentration or amount of guide RNA administered to the mouse relative to the concentration or amount of exogenous donor nucleic acid administered to the mouse. 43. The method of claim 34 wherein the altered variable in step (II) is an exogenous donor nucleic acid administered to the mouse. 44. A mouse for testing the ability of a CRISPR/Cas9 nuclease to modify a target genomic locus in vivo, containing a Cas9 expression cassette genomically integrated into the Rosa26 locus, wherein the Cas9 expression cassette contains a coding sequence for the Cas9 protein, wherein the Cas9 protein is expressed and contains the complete sequence shown in SEQ ID NO: 19. 45. The mouse of claim 44, wherein the Cas9 expression cassette further comprises a polyadenylation signal upstream of the coding sequence for the Cas9 protein, wherein the polyadenylation signal is flanked by recombinase recognition sites, and wherein the polyadenylation signal in the Cas9 expression cassette has been excised in a tissue-specific manner. 46. The mouse of claim 45, wherein the polyadenylation signal upstream of the coding sequence for the Cas9 protein was excised in the liver. 47. The mouse of claim 45 or 46, wherein the recombinase that recognizes recombinase recognition sites in the Cas9 expression cassette is Cre recombinase. 48. The mouse of claim 47, which further comprises a genomically integrated Cre recombinase expression cassette comprising a Cre recombinase coding sequence operably linked to a tissue-specific promoter. 49. A mouse according to claim 47 or 48, wherein the Cre recombinase gene is operably linked to an albumin promoter. 50. The mouse according to any one of paragraphs. 44-49, where the Cas9 expression cassette additionally contains a sequence encoding a fluorescent protein. 51. The mouse of claim 50, wherein the Cas9 expression cassette contains a multicistronic nucleic acid containing a coding sequence for the Cas9 protein and a fluorescent protein coding sequence separated by an internal ribosome entry intermediate site (IRES) or an intermediate 2A peptide coding sequence. 52. The mouse of claim 51, wherein the multicistronic nucleic acid in the Cas9 expression cassette contains a coding sequence for the Cas9 protein and a green fluorescent protein coding sequence separated by an intermediate P2A peptide coding sequence. 53. The mouse according to any one of paragraphs. 44-49, where the Cas9 expression cassette additionally does not contain a fluorescent protein coding sequence. 54. The mouse of any one of 44-53, wherein the Cas9 protein contains a protein tag. 55. The mouse of any one of 44-54, wherein the 5' end of the Cas9 expression cassette further comprises a 3' splicing sequence. 56. The mouse of any one of 44-55, wherein the Cas9 expression cassette is operably linked to an endogenous promoter. 57. The mouse according to any one of paragraphs. 44-55, where the Cas9 expression cassette is operably linked to an exogenous, constitutive promoter. 58. The mouse according to any one of paragraphs. 44-57, where the Cas9 expression cassette encodes a protein containing the sequence shown in SEQ ID NO: 13, 16 or 22. 59. The mouse of claim 58, wherein the Cas9 expression cassette contains the sequence shown in SEQ ID NO: 28, 29, 30, or 31. 60. The mouse of claim 59, wherein the Cas9 expression cassette contains the sequence shown in SEQ ID NOs: 1, 12, 14, 15, 17, 18, 20, or 21. 61. The mouse according to any one of paragraphs. 44-60, where the Cas9 expression cassette is integrated into the first intron of the Rosa26 locus. 62. A mouse according to any one of items 44-61 which is heterozygous for the Cas9 expression cassette. 63. The mouse according to any one of paragraphs. 44-61, which is homozygous for the Cas9 expression cassette. 64. The mouse according to any one of paragraphs. 44-63, where the Cas9 expression cassette is operably linked to the endogenous Rosa26 promoter, is inserted into the first intron of the Rosa26 locus and contains, from 5' to 3', the following: (i) 3' splicing sequence; and (ii) a coding sequence for the Cas9 protein. 65. Mouse cell for testing the ability of CRISPR/Cas9 nuclease to modify a target genomic locus, containing a Cas9 expression cassette genomically integrated into the Rosa26 locus, wherein the Cas9 expression cassette contains a coding sequence for the Cas9 protein, and wherein the Cas9 protein is expressed and contains the complete sequence shown in SEQ ID NO: 19. 66. The method of obtaining a mouse according to any one of paragraphs. 44-64, providing: (a) modifying the mouse embryonic stem (ES) cell genome to contain a Cas9 expression cassette genomically integrated into the Rosa26 locus, wherein the Cas9 expression cassette contains the coding sequence for the Cas9 protein, and wherein the Cas9 protein is expressed and contains the complete sequence shown in SEQ ID NO: 19; (b) identifying or selecting a genetically modified mouse ES cell containing the Cas9 expression cassette; (c) introducing a genetically modified mouse ES cell into a mouse host embryo; and (d) implantation and incubation of a mouse embryo host in a mouse surrogate mother. 67. The method of obtaining a mouse according to any one of paragraphs. 44-64, providing: (a) modification of the mouse embryonic genome at the single cell stage to contain a Cas9 expression cassette genomically integrated into the Rosa26 locus, wherein the Cas9 expression cassette contains the coding sequence for the Cas9 protein, and wherein the Cas9 protein is expressed and contains the complete sequence shown in SEQ ID NO: 19 ; (b) selecting a genetically modified mouse embryo at the single cell stage containing the Cas9 expression cassette; and (c) implantation and gestation of a genetically modified mouse embryo at the single cell stage in a mouse surrogate mother.

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