CN114292831B - Novel Cas enzyme and application - Google Patents

Abstract

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention provides a novel Cas enzyme, which has low homology with the reported Cas enzymes, can exhibit nuclease activity inside and outside the cell, and has broad application prospects.

Description

Novel Cas enzymes and their applications

Technical Field

The present invention relates to the field of gene editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to a novel Cas effector protein, a fusion protein comprising such a protein, and nucleic acid molecules encoding them. The present invention also relates to a complex and composition for nucleic acid editing (e.g., gene or genome editing), comprising a Cas protein or fusion protein of the present invention, or a nucleic acid molecule encoding them.

Background Art

CRISPR/Cas technology is a widely used gene editing technology that uses RNA to guide specific binding to target sequences on the genome and cut DNA to produce double-strand breaks, and uses biological non-homologous end joining or homologous recombination for site-specific gene editing.

The CRISPR/Cas9 system is the most commonly used Type II CRISPR system, which recognizes the PAM motif of 3’-NGG and performs blunt-end cleavage on the target sequence. The CRISPR/Cas Type V system is a newly discovered CRISPR system that has a 5’-TTN motif and performs sticky-end cleavage on the target sequence, such as Cpf1, C2c1, CasX, and CasY. However, the different CRISPR/Cas systems currently available have different advantages and disadvantages. For example, Cas9, C2c1, and CasX all require two RNAs for guide RNA, while Cpf1 only requires one guide RNA and can be used for multiple gene editing. CasX has a size of 980 amino acids, while the common Cas9, C2c1, CasY, and Cpf1 are usually around 1,300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are relatively complex and diverse, while C2c1 recognizes the rigorous 5’-TTN, so its target site is easier to predict than other systems, thereby reducing potential off-target effects.

In summary, given that the currently available CRISPR/Cas systems are limited by some defects, the development of a more robust new CRISPR/Cas system with multi-faceted good performance is of great significance to the development of biotechnology.

Summary of the invention

After a lot of experiments and repeated exploration, the inventors of the present application unexpectedly discovered a new type of nuclease (Cas enzyme). The Cas enzyme includes one or any of Cas-sf1, Cas-sf3, Cas-sf4, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10. Based on this discovery, the inventors have developed a new CRISPR/Cas system and a gene editing method and a nucleic acid detection method based on the system.

Cas effector proteins

On the one hand, the present invention provides a Cas protein (or, referred to as Cas enzyme), which is an effector protein in the CRISPR/Cas system, and the effector protein is selected from one or any several of Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10.

Among them, the amino acid sequences of Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10 are shown in SEQ ID No.1-7, respectively.

In one embodiment, the Cas protein amino acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID No.1-7, and substantially retains its biological function derived from the sequence.

In one embodiment, the Cas protein amino acid sequence has one or more amino acid substitutions, deletions or additions compared to any one of SEQ ID No.1-7, and substantially retains the biological function derived from the sequence; the one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions.

It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. As long as the substitution does not result in the inactivation of the biological activity of the protein, a conservative substitution in which an amino acid is replaced by other amino acids belonging to the same group falls within the scope of the present invention. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably generated by substitution according to Table 1. In addition, the present invention also covers proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.

Conservative amino acid substitutions can be performed at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be changed (deleted, substituted or replaced) without changing biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Amino acid substitutions can be performed in non-conserved regions of Cas enzymes. In general, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in conserved regions.

Table 1

Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

It is well known in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and/or C terminus of a protein while still retaining its functional activity. Therefore, proteins that have changed one or more amino acid residues from the N and/or C terminus of the Cas protein of the present invention while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification that changes or extends the protein coding sequence by including the amino acid coding sequence in the oligonucleotides used in the PCR amplification.

It should be recognized that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of Cas proteins can be prepared by mutations to DNA. It can also be accomplished by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling methods, combined with related screening methods, to perform single or multiple amino acid substitutions, deletions and/or insertions.

Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domain, lesser effects may be expected.

Those skilled in the art can identify the essential amino acids of the Cas protein according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of a bioinformatics system. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

In one embodiment, the Cas protein comprises an amino acid sequence shown in any one of SEQ ID No.1-7.

In one embodiment, the Cas protein is an amino acid sequence shown in any one of SEQ ID No.1-7.

In one embodiment, the Cas protein is a derivatized protein having the same biological function as a protein having a sequence shown in any one of SEQ ID No. 1-7.

The biological functions include, but are not limited to, the activity of binding to the guide RNA, the endonuclease activity, the activity of binding to and cutting a specific site of the target sequence under the guidance of the guide RNA, including but not limited to Cis cutting activity and Trans cutting activity.

The present invention also provides a fusion protein, which includes any one Cas protein selected from Cas-sf1, Cas-sf3, Cas-sf4, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10 and other modified parts.

In one embodiment, the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

In one embodiment, the modified portion is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting portion, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., a KRAB domain or a SID domain), a nuclease domain (e.g., Fok1), and a domain having an activity selected from the following: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity; and any combination thereof. The NLS sequence is well known to those skilled in the art, and examples thereof include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc and TUS protein.

In one embodiment, the NLS sequence is located at, near or close to a terminus (e.g., the N-terminus, the C-terminus, or both) of the Cas protein of the invention.

The epitope tag is well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art can select other suitable epitope tags (e.g., purification, detection or tracing).

The reporter gene sequence is well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

In one embodiment, the fusion protein of the present invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule, such as maltose binding protein (MBP), the DNA binding domain (DBD) of Lex A, the DBD of GAL4, and the like.

In one embodiment, the fusion protein of the invention comprises a detectable label, such as a fluorescent dye, such as FITC or DAPI.

In one embodiment, the Cas protein of the present invention is optionally coupled, conjugated or fused to the modification portion via a linker.

In one embodiment, the modification portion is directly linked to the N-terminus or C-terminus of the Cas protein of the present invention.

In one embodiment, the modified portion is connected to the N-terminus or C-terminus of the Cas protein of the present invention via a linker. Such linkers are well known in the art, and examples thereof include but are not limited to linkers comprising one or more (e.g., 1, 2, 3, 4 or 5) amino acids (e.g., Glu or Ser) or amino acid derivatives (e.g., Ahx, β-Ala, GABA or Ava), or PEG, etc.

The Cas protein, protein derivative or fusion protein of the present invention is not limited by the way it is produced. For example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

Cas protein nucleic acid

On the other hand, the present invention provides an isolated polynucleotide comprising: a polynucleotide sequence encoding the Cas protein or fusion protein of the present invention.

In one embodiment, the polynucleotide sequence is codon optimized for expression in prokaryotes. In one embodiment, the polynucleotide sequence is codon optimized for expression in eukaryotic cells.

In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

Direct Repeat Sequence

On the other hand, the present invention provides an engineered co-directional repeat sequence that forms a complex with any one Cas protein selected from the above-mentioned Cas-sf1, Cas-sf3, Cas-sf4, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10.

The direct repeat sequence is connected to a guide sequence that can hybridize with the target sequence to form a guide RNA (guideRNA or gRNA).

The hybridization of the target sequence with the gRNA represents at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity between the nucleic acid sequences of the target sequence and the gRNA, so that they can hybridize to form a complex; or represents that the nucleic acid sequences of the target sequence and the gRNA have at least 12, 15, 16, 17, 18, 19, 20, 21, 22, or more bases that can complementarily pair to form a complex.

In some embodiments, the direct repeat sequence has at least 90% sequence identity with any one of SEQ ID No.8-14, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In some embodiments, the direct repeat sequence has a sequence having one or more base substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID No.8-14.

In some embodiments, the direct repeat sequence is shown in any one of SEQ ID No. 8-14, or in any one of SEQ ID No. 16-22.

In the present invention, SEQ ID No.8-14 correspond to the prototype direct repeat sequences of Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9 and Cas-sf10, respectively; SEQ ID No.16-22 correspond to the mature direct repeat sequences of Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9 and Cas-sf10, respectively.

Guide RNA (gRNA)

On the other hand, the present invention provides a gRNA, which includes a first segment and a second segment; the first segment is also called a "skeleton region", "protein binding segment", "protein binding sequence", or "Direct Repeat sequence"; the second segment is also called a "targeting sequence for targeting nucleic acid" or "targeting segment for targeting nucleic acid", or "guide sequence for targeting target sequence".

The first segment of the gRNA is capable of interacting with the Cas protein of the present invention, thereby forming a complex between the Cas protein and the gRNA.

The targeting sequence of the targeting nucleic acid of the present invention or the targeting section of the targeting nucleic acid comprises a nucleotide sequence complementary to the sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid of the present invention or the targeting section of the targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the targeting sequence of the targeting nucleic acid or the targeting section of the targeting nucleic acid can be changed, or can be modified to hybridize any desired sequence in the target nucleic acid. The nucleic acid is selected from DNA or RNA.

The percent complementarity between the targeting sequence of a targeting nucleic acid or the targeting segment of a targeting nucleic acid and the target sequence of a target nucleic acid 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 "skeleton region", "protein binding segment", "protein binding sequence", or "direct repeat sequence" of the gRNA of the present invention can interact with the CRISPR protein (or, Cas protein). The gRNA of the present invention guides the interacting Cas protein to a specific nucleotide sequence in the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

Preferably, the guide RNA comprises a first segment and a second segment from the 5' to the 3' direction.

In the present invention, the second segment can also be understood as a guide sequence that hybridizes with the target sequence.

The gRNA of the present invention can form a complex with the Cas protein.

Carrier

The present invention also provides a vector, which comprises the Cas protein, isolated nucleic acid molecule or polynucleotide as described above; preferably, it also includes a regulatory element operably linked thereto.

In one embodiment, the regulatory element is selected from one or more of the following groups: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, marker gene.

In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

In some embodiments, the vector included in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

Vector system

The present invention provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, which includes a Cas protein or a nucleic acid sequence encoding the Cas protein and a nucleic acid encoding one or more guide RNAs.

In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.

In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs do not naturally co-exist.

The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize with the genomic loci of the DNA molecule encoding the one or more gene products, and guide the Cas protein to the genomic loci of the DNA molecule encoding the one or more gene products. After the Cas protein reaches the target sequence position, it modifies, edits or cuts the target sequence, thereby changing or modifying the expression of the one or more gene products.

The cells of the present invention include one or more of animals, plants or microorganisms.

In some embodiments, the Cas protein is codon optimized for expression in a cell.

In some embodiments, the Cas protein directs cleavage of one or both strands at the location of the target sequence.

The present invention also provides an engineered non-naturally occurring vector system, which may include one or more vectors, wherein the one or more vectors include:

a) a first regulatory element, which is operably linked to the gRNA,

b) a second regulatory element, which is operably linked to the Cas protein;

Components (a) and (b) are located on the same or different carriers of the system.

The first and second regulatory elements include a promoter (e.g., a constitutive promoter or an inducible promoter), an enhancer (e.g., a 35S promoter or a 35S enhanced promoter), an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).

In some embodiments, the vector in the system is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector), and can also be a plasmid, a virus, a cosmid, a phage, etc., which are well known to those skilled in the art.

In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microbubble, and a gene gun.

In one embodiment, when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence shown as TTN, wherein N is selected from A, G, T, and C.

In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., an organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

In one embodiment, the Cas protein is connected to one or more NLS sequences. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is connected to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

On the other hand, the present invention relates to an engineered CRISPR system, which comprises the above-mentioned Cas protein and one or more guide RNAs, wherein the guide RNA includes a direct repeat sequence and a spacer sequence capable of hybridizing with a target nucleic acid, and the Cas protein can bind to the guide RNA and target a target nucleic acid sequence complementary to the spacer sequence.

Protein-nucleic acid complexes/compositions

In another aspect, the present invention provides a compound or composition comprising:

(i) a protein component selected from: the above-mentioned Cas protein, derivatized protein or fusion protein, and any combination thereof; and

(ii) a nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to a Cas protein of the present invention.

The protein component and the nucleic acid component are combined with each other to form a complex.

In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and/or, the second component is non-naturally occurring or modified.

Activated CRISPR complex

On the other hand, the present invention also provides an activated CRISPR complex, the activated CRISPR complex comprising: (1) a protein component selected from: a Cas protein, a derivatized protein or a fusion protein of the present invention, and any combination thereof; (2) a gRNA comprising (a) a guide sequence capable of hybridizing with a target sequence; and (b) a direct repeat sequence capable of binding to the Cas protein of the present invention; and (3) a target sequence bound to the gRNA. Preferably, the binding is carried out by binding the targeting sequence of the targeting nucleic acid on the gRNA to the target nucleic acid.

The terms "activated CRISPR complex", "activated complex" or "ternary complex" used in this article refer to the complex after the Cas protein, gRNA and target nucleic acid in the CRISPR system are bound or modified.

The Cas protein and gRNA of the present invention can form a binary complex, which is activated when bound to a nucleic acid substrate to form an activated CRISPR complex. The nucleic acid substrate is complementary to the spacer sequence in the gRNA (or referred to as a guide sequence that hybridizes with the target nucleic acid). In some embodiments, the spacer sequence of the gRNA completely matches the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (continuous or discontinuous) of the target substrate.

In a preferred embodiment, the activated CRISPR complex may exhibit collateral nuclease cleavage activity, which refers to the non-specific cleavage activity or random cleavage activity of the activated CRISPR complex on single-stranded nucleic acids, also known as trans cleavage activity in the art.

Delivery and delivery compositions

The Cas protein, gRNA, fusion protein, nucleic acid molecule, vector, system, complex and composition of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, nuclear transfection, magnetofection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, etc.

Therefore, in another aspect, the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: the Cas protein, fusion protein, nucleic acid molecule, vector, system, complex and composition of the present invention.

In one embodiment, the delivery vehicle is a particle.

In one embodiment, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

Host cells

The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, which comprises: the Cas protein, fusion protein, nucleic acid molecule, protein-nucleic acid complex, activated CRISPR complex, vector, and delivery composition of the present invention.

In certain embodiments, the cell is a prokaryotic cell.

In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, such as a cell of a non-human primate, a cow, a sheep, a pig, a dog, a monkey, a rabbit, a rodent (such as a rat or a mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (such as a chicken), a fish or a crustacean (such as a clam, a shrimp). In certain embodiments, the cell is a plant cell, such as a cell or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, such as algae, tree or production plant, fruit or vegetable (for example, trees such as citrus trees, nut trees; Solanum, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

In certain embodiments, the cell is a stem cell or a stem cell line.

In certain cases, the host cells of the invention contain genetic or genomic modifications that are not present in their wild type.

Gene Editing Methods and Applications

The Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, activated CRISPR complex or host cell of the present invention can be used for any one or more of the following purposes: targeting and/or editing target nucleic acid; cutting double-stranded DNA, single-stranded DNA or single-stranded RNA; non-specific cutting and/or degradation of side branch nucleic acid; non-specific cutting of single-stranded nucleic acid; nucleic acid detection; detection of nucleic acid in target sample; specific editing of double-stranded nucleic acid; base editing of double-stranded nucleic acid; base editing of single-stranded nucleic acid. In other embodiments, it can also be used to prepare reagents or kits for any one or more of the above purposes.

The present invention also provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition or activated CRISPR complex in gene editing, gene targeting or gene cutting; or, use in the preparation of reagents or kits for gene editing, gene targeting or gene cutting.

In one embodiment, the gene editing, gene targeting or gene cleavage is performed inside and/or outside the cell.

The present invention also provides a method for editing a target nucleic acid, targeting a target nucleic acid, or cutting a target nucleic acid, the method comprising contacting the target nucleic acid with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned vector system, the above-mentioned delivery composition, or the above-mentioned activated CRISPR complex. In one embodiment, the method is to edit the target nucleic acid, target the target nucleic acid, or cut the target nucleic acid in a cell and/or outside the cell.

The gene editing or editing of target nucleic acid includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and/or inserting polynucleotides, gene mutations.

The editing can be performed in prokaryotic cells and/or eukaryotic cells.

On the other hand, the present invention also provides the use of the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in nucleic acid detection, or in the preparation of a reagent or kit for nucleic acid detection.

On the other hand, the present invention also provides a method for cutting single-stranded nucleic acids, the method comprising contacting a nucleic acid population with the above-mentioned Cas protein and gRNA, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, and the Cas protein cuts the plurality of non-target single-stranded nucleic acids.

The gRNA is capable of binding to the Cas protein.

The gRNA is capable of targeting the target nucleic acid.

The contacting can be inside a cell in vitro, ex vivo or in vivo.

Preferably, the cleavage of the single-stranded nucleic acid is non-specific cleavage.

On the other hand, the present invention also provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition or activated CRISPR complex in non-specific cleavage of single-stranded nucleic acid, or in the preparation of a reagent or kit for non-specific cleavage of single-stranded nucleic acid.

On the other hand, the present invention also provides a kit for gene editing, gene targeting or gene cutting, which comprises the above-mentioned Cas protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned vector system, the above-mentioned delivery composition, the above-mentioned activated CRISPR complex or the above-mentioned host cell.

On the other hand, the present invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising: (a) a Cas protein, or a nucleic acid encoding the Cas protein; (b) a guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA; and (c) a single-stranded nucleic acid detector that is single-stranded and does not hybridize with the guide RNA.

It is known in the art that the precursor RNA can be cleaved or processed into the mature guide RNA described above.

On the other hand, the invention provides the use of the above-mentioned Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, activated CRISPR complex or host cell in the preparation of a preparation or a kit, wherein the preparation or kit is used for:

(i) gene or genome editing;

(ii) target nucleic acid detection and/or diagnosis;

(iii) editing a target sequence in a target locus to modify an organism or non-human organism;

(iv) treatment of disease;

(v) targeting target genes;

(vi) Cutting the target gene.

Preferably, the above-mentioned gene or genome editing is performed inside or outside the cell.

Preferably, the target nucleic acid detection and/or diagnosis is performed in vitro.

Preferably, the treatment of the disease is the treatment of a condition caused by a defect in the target sequence in the target locus.

In another aspect, the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, gRNA (guide RNA) and a single-stranded nucleic acid detector, the gRNA comprising a region binding to the Cas protein and a guide sequence hybridizing with the target nucleic acid; detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize with the gRNA.

Method for specifically modifying target nucleic acid

On the other hand, the present invention also provides a method for specifically modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex.

The specific modification can occur in vivo or in vitro.

The specific modification can occur inside or outside the cell.

In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, for example, an animal cell, a plant cell, or a microbial cell.

In one embodiment, the modification refers to a break in the target sequence, such as a single-strand/double-strand break in DNA, or a single-strand break in RNA.

In some cases, the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target nucleic acid.

In one embodiment, the modification further comprises inserting an editing template (eg, an exogenous nucleic acid) into the break.

In one embodiment, the method further comprises: contacting the editing template with the target nucleic acid, or delivering it to a cell comprising the target nucleic acid. In this embodiment, the method repairs the broken target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation, including insertion, deletion, or substitution of one or more nucleotides of the target gene, and in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

Detection (non-specific cleavage)

On the other hand, the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, thereby detecting the target nucleic acid.

In the present invention, the target nucleic acid includes ribonucleotides or deoxyribonucleotides; including single-stranded nucleic acids, double-stranded nucleic acids, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA. In one embodiment, the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water, human body, animals, plants, etc.

Preferably, the target nucleic acid is a product enriched or amplified by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM, etc.

In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or a SNP site or a nucleic acid that differs from a control; preferably, the virus is a plant virus or an animal virus, for example, a papillomavirus, a hepadnavirus, a herpes virus, an adenovirus, a poxvirus, a parvovirus, a coronavirus; preferably, the virus is a coronavirus, preferably, SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

In some embodiments, the target nucleic acid is derived from cells, for example, from cell lysate.

In one embodiment, the target nucleic acid includes DNA, RNA, preferably single-stranded nucleic acid or double-stranded nucleic acid or nucleic acid modification.

In the present invention, the gRNA has at least 50% matching degree with the target sequence on the target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

In one embodiment, when the target sequence contains one or more characteristic sites (such as a specific mutation site or SNP), the characteristic sites are completely matched with the gRNA.

In one embodiment, the detection method may include one or more gRNAs with different guide sequences, which target different target sequences.

In the present invention, the single-stranded nucleic acid detector includes but is not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifiers, and single-stranded nucleic acid detectors containing a base-free spacer, etc.; "nucleic acid analogs" include but are not limited to: locked nucleic acid, bridge nucleic acid, morpholino nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2'oxymethyl RNA, 2'methoxyacetyl RNA, 2'fluoro RNA, 2'amino RNA, 4'thio RNA and combinations thereof, including optional ribonucleotides or deoxyribonucleotide residues.

In the present invention, the detectable signal is realized by the following means: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence detection, colloidal phase change/dispersion, electrochemical detection and semiconductor-based detection.

In the present invention, preferably, a fluorescent group and a quenching group are respectively arranged at both ends of the single-stranded nucleic acid detector, and a detectable fluorescent signal can be exhibited when the single-stranded nucleic acid detector is cut. The fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In other embodiments, different labeling molecules are respectively set at the 5' end and the 3' end of the single-stranded nucleic acid detector, and the colloidal gold test results of the single-stranded nucleic acid detector before and after being cut by the Cas protein are detected by colloidal gold detection; the single-stranded nucleic acid detector will show different color development results on the colloidal gold detection line and the quality control line before and after being cut by the Cas protein.

In some embodiments, the method of detecting a target nucleic acid may further include comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.

In some embodiments, the method of detecting a target nucleic acid can also include using an RNA reporter nucleic acid and a DNA reporter nucleic acid (e.g., fluorescent color) on different channels, and determining the level of the detectable signal by measuring the signal levels of the RNA and DNA reporter molecules, and by measuring the amount of the target nucleic acid in the RNA and DNA reporter molecules, and sampling based on the combined (e.g., using a minimum or product) level of the detectable signal.

In one embodiment, the target nucleic acid is present within a cell.

In one embodiment, the cell is a prokaryotic cell.

In one embodiment, the cell is a eukaryotic cell.

In one embodiment, the cell is an animal cell.

In one embodiment, the cell is a human cell.

In one embodiment, the cell is a plant cell, such as a cell from a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

In one embodiment, the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro.

In one embodiment, the target gene is present in a plasmid.

Definition of terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

Cas proteins

In the present invention, Cas protein, Cas enzyme, and Cas effector protein can be used interchangeably; the inventors have discovered and identified for the first time a Cas effector protein having an amino acid sequence selected from the following:

(i) a sequence shown in any one of SEQ ID No. 1-7;

(ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID No. 1-7; or

(iii) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID Nos. 1-7.

Nucleic acid cleavage or nucleic acid cleavage herein includes: DNA or RNA breakage (Cis cleavage) in the target nucleic acid produced by the Cas enzyme described herein, DNA or RNA breakage in the side branch nucleic acid substrate (single-stranded nucleic acid substrate) (i.e., non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.

CRISPR system

As used herein, the terms "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning generally understood by those skilled in the art, which generally includes transcription products or other elements related to the expression of CRISPR-associated ("Cas") genes, or transcription products or other elements capable of directing the activity of the Cas genes.

CRISPR/Cas complex

As used herein, the term "CRISPR/Cas complex" refers to a complex formed by the binding of a guide RNA or mature crRNA to a Cas protein, which comprises a direct repeat sequence that hybridizes to a guide sequence of a target sequence and binds to a Cas protein, and the complex is capable of recognizing and cleaving a polynucleotide that can hybridize to the guide RNA or mature crRNA.

Guide RNA (gRNA)

As used herein, the terms "guide RNA (gRNA)", "mature crRNA", "guide sequence" are used interchangeably and have meanings generally understood by those skilled in the art. In general, the guide RNA may comprise a direct repeat sequence and a guide sequence, or may consist essentially of or consist of a direct repeat sequence and a guide sequence.

In some cases, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the capabilities of ordinary technicians in this field. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

Target sequence

"Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, such as a sequence having complementarity with the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR/Cas complex (including Cas protein and gRNA). Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR/Cas complex.

The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

In the present invention, "target sequence" or "target polynucleotide" or "target nucleic acid" can be any endogenous or exogenous polynucleotide to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-200 nucleotides, preferably 2-150 nucleotides, preferably 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, more preferably 5-15 nucleotides, preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

The single-stranded nucleic acid detector includes different reporting groups or labeling molecules at both ends. When it is in the initial state (i.e., uncleaved state), it does not present a reporting signal. When the single-stranded nucleic acid detector is cut, it presents a detectable signal, i.e., there is a detectable difference between after cutting and before cutting.

In one embodiment, the reporter group or labeling molecule includes a fluorescent group and a quencher group, the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quencher group is selected from one or any several of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In one embodiment, the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5' end and a second molecule (such as biotin) connected to the 3' end. The reaction system containing the single-stranded nucleic acid detector is used to detect the target nucleic acid in conjunction with the flow strip (preferably, colloidal gold detection method). The flow strip is designed to have two capture lines, with an antibody that binds to the first molecule (i.e., the first molecule antibody) at the sample contact end (colloidal gold), an antibody that binds to the first molecule antibody at the first line (control line), and an antibody that binds to the second molecule (i.e., the second molecule antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecule antibody binds to the first molecule and carries the cut or uncut oligonucleotide to the capture line, and the cut reporter will bind to the antibody of the first molecule antibody at the first capture line, and the uncut reporter will bind to the second molecule antibody at the second capture line. The binding of the reporter group to each line will result in a strong readout/signal (e.g., color). As more reporters are cut, more signals will accumulate at the first capture line, and less signals will appear at the second line. In some aspects, the present invention relates to the use of a flow strip as described herein for detecting nucleic acids. In some aspects, the present invention relates to a method for detecting nucleic acids using a flow strip as defined herein, such as a (lateral) flow test or a (lateral) flow immunochromatographic assay. In some aspects, the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the position of the molecules can be changed, as long as the reporting principle is the same or similar to the present invention, the improved method is also included in the present invention.

The detection method of the present invention can be used for quantitative detection of target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal strength of the reporter group, such as according to the luminescence intensity of the fluorescent group, or according to the width of the color band.

Wild type

As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, which refers to the typical form of an organism, strain, gene, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

Derivatization

As used herein, the term "derivatization" refers to a chemical modification of an amino acid, polypeptide or protein wherein one or more substituents have been covalently attached to the amino acid, polypeptide or protein. The substituents may also be referred to as side chains.

A derivatized protein is a derivative of the protein. Generally, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., the activity of binding to the guide RNA, the endonuclease activity, the activity of binding to and cutting a specific site of the target sequence under the guidance of the guide RNA), that is, the derivative of the protein has the same activity as the protein.

Derivatized Protein

Also known as "protein derivatives", refers to modified forms of proteins, for example, wherein one or more amino acids of the protein may be deleted, inserted, modified and/or substituted.

Non-naturally occurring

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human effort. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is associated in nature or as found in nature.

Orthologue (ortholog)

As used herein, the term "orthologue" has the meaning commonly understood by those skilled in the art. As a further guide, an "orthologue" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as its orthologue.

Identity

As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Carrier

The term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. The vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site.

One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

Another type of vector is a viral vector, wherein a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced.

Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are replicated together with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."

Host cells

As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.

Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, and the like.

Regulatory elements

As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements include those sequences that direct the constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct the nucleotide sequence to be expressed only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

Promoter

As used herein, the term "promoter" has a meaning well known to those skilled in the art, and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell essentially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a gene product in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

NLS

A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the nucleus by nuclear transport, i.e., a protein with an NLS is transported to the nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, NLSs from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises a PKKKRKV sequence. In some embodiments, the NLS comprises an AVKRPAATKKAGQAKKKKLD sequence. In some embodiments, the NLS comprises a PAAKRVKLD sequence. In some embodiments, the NLS comprises an MSRRRKANPTKLSENAKKLAKEVEN sequence. In some embodiments, the NLS comprises a KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequence KIPIK in the yeast transcription repressor Matα2, and PY-NLS.

Operatively connected

As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

Complementarity

As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick or other non-traditional types. The percentage of complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

Strict conditions

As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence predominantly hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

Hybridization

The terms "hybridize" or "complementary" or "substantially complementary" refer to a nucleic acid (e.g., RNA, DNA) comprising a nucleotide sequence that enables it to non-covalently bind, i.e., form base pairs and/or G/U base pairs, "anneal" or "hybridize" with another nucleic acid in a sequence-specific, anti-parallel manner (i.e., nucleic acids specifically bind to complementary nucleic acids).

Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, which are variables well known in the art. Typically, the length of a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

It should be understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. A polynucleotide may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to the target region in the target nucleic acid sequence with which it hybridizes.

The hybridization of the target sequence and the gRNA means that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the nucleic acid sequences of the target sequence and the gRNA can hybridize to form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the nucleic acid sequences of the target sequence and the gRNA can complement each other and hybridize to form a complex.

Express

As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

Connectors

As used herein, the term "joint" refers to a linear polypeptide formed by connecting a plurality of amino acid residues via peptide bonds. The joint of the present invention can be an artificially synthesized amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such joint polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J. et al. (1994) Structure 2: 1121-1123).

treat

As used herein, the term "treat" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and/or delaying the progression of a disorder.

Subjects

As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

animal

For example, mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) suffers from a disorder (e.g., a disorder caused by a disease-related gene defect).

plant

The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., melon, watermelon, Crenshaw melon, honeydew melon, cantaloupe), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, bulb onion, turnip, cabbage, eggplant (also known as eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and/or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, Currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, silver grass, corn/maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oil plants (rapeseed, mustard cabbage, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plant; and/or bedding plants, such as flowering plants, cacti, succulent plants and/or ornamental plants, and trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

Advantageous Effects of the Invention

The present invention discovered a new type of Cas enzyme, which can exhibit nuclease activity in vivo and in vitro and has broad application prospects.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Experimental results of in vitro cleavage activity of different Cas proteins.

Figure 2. Editing efficiency of Cas-sf4 and Cas-sf1 in protoplasts.

Figure 3. Types of gene editing by Cas-sf4 and Cas-sf1 in protoplasts.

Figure 4. Fluorescence results of Cas-sf1 used for in vitro nucleic acid detection.

Figure 5. Fluorescence results of Cas-sf4 used for in vitro nucleic acid detection.

Figure 6. Fluorescence results of Cas-sf8 used for in vitro nucleic acid detection.

Figure 7. Fluorescence results of Cas-sf9 used for in vitro nucleic acid detection.

Figure 8. Fluorescence results of Cas-sf10 used for in vitro nucleic acid detection.

Sequence information

DETAILED DESCRIPTION

The following examples are only used to describe the present invention, but not to limit the present invention. Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to the conventional methods well known in the art and described in various references. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual" (MOLECULAR CLONING: A LABORATORY MANUAL), 2nd edition (1989); "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY" (F.M. Ausubel et al., ed., (1987)); "METHODS IN ENZYMOLOGY" series (Academic Publishing Company): "PCR 2: A PRACTICAL METHOD" (PCR 2: A PRACTICAL METHOD) APPROACH) (M. J. MacPherson, B. D. Hames, and G. R. Taylor, eds. (1995)), ANTIBODIES, A LABORATORY MANUAL, Harlow and Lane, eds. (1988), and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

Example 1. Acquisition of Cas protein

The inventors analyzed the metagenome of uncultured organisms and identified a new class of Cas enzymes through deredundancy and protein clustering analysis, which were named Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10, respectively. Their amino acid sequences are shown in SEQ ID No.1-7, respectively. Blast results showed that the sequence consistency of this Cas protein was low with that of the reported Cas proteins.

Analysis found that the prototype direct repeat sequences of gRNA corresponding to Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10 are shown as SEQ ID No.8-14, respectively, and the corresponding PAM is TTN (N can be any base); the corresponding mature direct repeat sequences are shown as SEQ ID No.16-22, respectively.

Example 2. Verification of Cas protein cleavage activity in vitro

Cas-sf4, Cas-sf1, Cas-sf3, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10 proteins were respectively constructed into the pet30a expression vector, transferred into Escherichia coli, and purified prokaryotic expressed proteins were performed to obtain purified target proteins.

1 ug of purified Cas protein, 500 ng of in vitro transcribed gRNA, and 300 ng of PCR product with PAM as TTC were incubated at 37°C for 1 h or overnight digestion. The sequence of the PCR product is shown in SEQ ID No. 15; the gRNA sequences for different Cas proteins are shown in Table 1.

Table 1. gRNA sequences used in in vitro cleavage experiments

The results are shown in Figure 1. The arrows in the figure indicate PCR products. As shown in Figure 1, different Cas proteins cut PCR products to varying degrees, especially Cas-sf4, which has the highest efficiency in cutting PCR products.

Example 2. Editing efficiency of Cas proteins in corn protoplasts

In order to verify whether the above Cas proteins can produce editing effects in eukaryotic cells. First, plasmids expressing different Cas proteins and crRNA plasmids were constructed using TTTC's PAM to transform corn protoplasts. After transformation, DNA was extracted, fragments containing the target site were amplified, and second-generation sequencing was performed; the above-mentioned targeted corn gene is: SBE2.2 (Zm00001d003817), and the gRNA sequences for different Cas proteins are shown in Table 2.

Table 2. gRNA sequences used in maize protoplasts

The test results show that Cas-sf4 and Cas-sf1 can show obvious editing efficiency in corn protoplasts. As shown in Figure 2, the editing efficiency of Cas-sf1 in corn protoplasts is 2.4%, and the editing efficiency of Cas-sf4 in corn protoplasts is 0.7%. The editing types of Cas-sf4 and Cas-sf1 against SBE2.2 are shown in Figure 3.

Example 3. Application of Cas protein in in vitro nucleic acid detection

This example verifies the trans-cleavage activity of the Cas enzyme by in vitro testing. In this example, a gRNA that can be paired with a target nucleic acid is used to guide the Cas enzyme to recognize and bind to the target nucleic acid; then, the Cas enzyme stimulates the trans-cleavage activity of any single-stranded nucleic acid, thereby cutting the single-stranded nucleic acid detector in the system; a fluorescent group and a quenching group are respectively set at both ends of the single-stranded nucleic acid detector, and if the single-stranded nucleic acid detector is cut, fluorescence will be stimulated; in other embodiments, the two ends of the single-stranded nucleic acid detector can also be set as a label that can be detected by colloidal gold.

In this embodiment, the target nucleic acid is selected as a single-stranded DNA, N-B-i3g1-ssDNA0, and its sequence is: cgacattccgaagaacgctgaagcgctgggggcaaattgtgcaatttgcggc.

The 5' to 3' ends of the gRNA are the DR regions of different Cas proteins and the sequences of the target nucleic acid, and the sequence of the target nucleic acid is cccccagcgcuucagcguuc;

The sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1.

The following reaction system was used: the final concentration of Cas enzyme was 50 nM, the final concentration of gRNA was 50 nM, the final concentration of target nucleic acid was 500 nM, and the final concentration of single-stranded nucleic acid detector was 200 nM. Incubate at 37°C and read FAM fluorescence/1 min. No target nucleic acid was added to the control group.

In this embodiment, the trans-cutting activity of Cas-sf4, Cas-sf1, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10 was tested, and the DR region of the gRNA selected the mature direct repeat sequence of the corresponding protein. The mature direct repeat sequences of Cas-sf4, Cas-sf1, Cas-sf6, Cas-sf8, Cas-sf9, and Cas-sf10 proteins are shown in SEQ ID No. 18, 19, 21, 22, 23, and 24, respectively. Among them, no obvious trans-cutting activity was detected for Cas-sf6. As shown in Figures 4-8, compared with the control without adding target nucleic acid, in the presence of target nucleic acid, Cas-sf4, Cas-sf1, Cas-sf8, Cas-sf9, and Cas-sf10 can cut the single-stranded nucleic acid in the system and quickly report fluorescence. The above experiments show that, in combination with single-stranded nucleic acid detectors, Cas-sf4, Cas-sf1, Cas-sf8, Cas-sf9, and Cas-sf10 can be used for the detection of target nucleic acids. In Figures 4-8, line 1 is the experimental result of adding target nucleic acids, and line 2 is the control group without adding target nucleic acids.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

SEQUENCE LISTING

<110> Shandong Shunfeng Biotechnology Co., Ltd.

<120> Novel Cas enzymes and their applications

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Met Ile Lys Met Met Lys Glu Lys Ser Ile Trp Asn Glu Phe Thr Asn

1 5 10 15

Met Tyr Ser Ile Ser Lys Thr Leu Arg Phe Lys Leu Lys Pro Ile Gly

20 25 30

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

35 40 45

Asp Arg Glu Lys Gly Phe Asn Asn Ile Lys Lys Ile Met Asp Asp Tyr

50 55 60

Tyr Arg Tyr Phe Ile Glu Lys Cys Leu Asn Gly Ile Lys Leu Glu Lys

65 70 75 80

Lys Asp Leu Glu Ala Tyr Gln Lys Val Tyr Glu Asp Leu Lys Lys Asp

85 90 95

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

100 105 110

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

115 120 125

Gln Leu Phe Lys Lys Glu Leu Ile Thr His Ile Leu Pro Glu Trp Leu

130 135 140

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

145 150 155 160

Asn Trp Ser Thr Tyr Phe Thr Gly Phe Phe Asn Asn Arg Lys Asn Val

165 170 175

Phe Ser Glu Lys Glu Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val His

180 185 190

Val Asn Leu Pro Lys Tyr Leu Asp Asn Val Ser Arg Phe Glu Lys Ile

195 200 205

Lys Glu Phe Asn Leu Asp Leu Lys Thr Leu Glu Asn Asp Phe Lys Asp

210 215 220

Val Leu Asp Asn Met Asp Leu Asn Glu Phe Phe Ser Val Asn Asn Phe

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Asp Ser Glu Ser Ser Ser Phe Val Ile Glu Lys Phe Lys Asp Lys Lys

305 310 315 320

Glu Ile Phe Glu Thr Ile Asp Val Phe Tyr Lys Glu Phe Asn Lys Tyr

325 330 335

Ser Ser Lys Ile Lys Glu Ser Ile Thr Lys Leu Asn Asn Cys Asp Ser

340 345 350

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

355 360 365

Gly Leu Phe Asn Asp Trp Asn Lys Ile Asp Gly Gly Leu Arg Arg His

370 375 380

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

385 390 395 400

Lys Glu Leu Asp Lys Cys Met Lys Ser Lys Tyr Phe Ser Leu Tyr Glu

405 410 415

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

420 425 430

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

435 440 445

Asp Leu Phe Glu Asn Ile Lys Ser Lys Tyr Ser Glu Phe Asn Lys Ile

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Asp Arg Asn Lys Thr Thr Lys Leu Thr Glu Lys Ser Ser Glu Asn Asp

465 470 475 480

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

485 490 495

Phe Ile Lys Pro Leu His Leu Asn Phe Lys Lys Asn Glu Asp Glu Lys

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Gly Ser Asn Ala Leu Glu Thr Asp Ser Asp Phe Tyr Asn Tyr Phe Asn

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

530 535 540

Arg Asn Tyr Val Thr Gln Lys Pro Phe Ser Thr Lys Lys Phe Lys Leu

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

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

580 585 590

Gly Ile Ile Asp Lys Asn Asn Thr Lys Ile Phe Leu Asn Gln Gln Asn

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Ser Asn Ser Ser Val Val Tyr Glu Lys Leu Cys Tyr Lys Leu Val Ser

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

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

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Glu His Lys Lys Gly Asn Thr Phe Ser Ile Glu Ser Cys His Lys Leu

660 665 670

Ile Asp Tyr Phe Lys Glu Cys Met Pro Asn Tyr Lys Pro Asn Pro Asn

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Asp Lys Tyr Gly Trp Asp Val Phe Lys Phe Lys Phe Ser Asp Thr Lys

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

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Tyr Lys Ile Trp Phe Glu Asn Ile Asp Glu Ser Tyr Leu Asn Lys Leu

725 730 735

Val Asp Glu Gly Lys Leu Tyr Leu Phe Gln Ile Trp Asn Lys Asp Phe

740 745 750

Ser Lys Tyr Ser Lys Gly Lys Pro Asn Leu His Thr Met Tyr Trp Lys

755 760 765

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

770 775 780

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

785 790 795 800

Thr His Pro Lys Asn Ile Ser Ile Asp Asn Lys Asn Pro Ile Lys Asn

805 810 815

Lys Glu Lys Ser Thr Phe Asn Tyr Asp Leu Ile Lys Asn Lys Arg Tyr

820 825 830

Ser Glu Asp Ser Phe Met Phe His Cys Pro Ile Thr Leu Asn Phe Lys

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Ala Lys Asp Gln Ser Lys Ser Ile His Lys Leu Val Asn Lys Phe Ile

850 855 860

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

865 870 875 880

Arg Asn Leu Ala Tyr Tyr Thr Leu Val Asn Ser Asp Gly Asn Ile Ile

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

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Asp Tyr Gln Glu Lys Leu Asp Gln Ile Glu Gly Asp Arg Asp Lys Ala

915 920 925

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

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Tyr Leu Ser Gln Val Ile His Lys Ile Ser Lys Leu Val Ile Glu His

945 950 955 960

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

965 970 975

Arg Phe Lys Ile Glu Lys Gln Ile Tyr Gln Lys Phe Glu Lys Met Leu

980 985 990

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

995 1000 1005

Ser Gly Gly Asn Leu Asn Ala Tyr Gln Leu Thr Asn Lys Phe Glu

1010 1015 1020

Ser Phe Gln Lys Leu Gly Lys Gln Ser Gly Ile Ile Tyr Tyr Val

1025 1030 1035

Asp Ala Tyr Lys Thr Ser Lys Ile Cys Pro Lys Thr Gly Phe Val

1040 1045 1050

Asn Leu Leu Tyr Pro Lys Phe Glu Asn Ile Leu Lys Ser Gln Glu

1055 1060 1065

Phe Ile Lys Lys Phe Lys Ser Ile Lys Tyr His Lys Asp Glu Asp

1070 1075 1080

Leu Phe Glu Phe Asn Phe Asn Tyr Ser Asp Phe Lys Lys Asp Gln

1085 1090 1095

Lys Glu Lys Leu Glu Gln Asp Asn Trp Ser Ile Trp Ser Asn Gly

1100 1105 1110

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

1115 1120 1125

Thr Thr Lys Glu Phe Lys Val Thr Glu Lys Leu Lys Glu Leu Phe

1130 1135 1140

Glu Asn His Asn Ile Asp Tyr Asn Ser Gly Asn Asp Leu Ile Glu

1145 1150 1155

Gln Ile Val Thr Ile Glu Asn Lys Ser Phe Tyr Glu Ser Leu Ile

1160 1165 1170

Tyr Ile Leu Lys Ile Ile Leu Lys Leu Arg Asn Ser Tyr Ser Asp

1175 1180 1185

Phe Glu Val Lys Gln Phe Lys Lys Lys Leu Gly Asn Lys Phe Lys

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Asp Ala Asp Ala Asn Gly Ala Phe His Ile Ala Leu Lys Gly Leu

1235 1240 1245

Met Val Ile Asp Lys Ile Lys Lys Phe Asp Asp Val Asp Glu Lys

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

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Val Val Lys Arg Ile Asn Arg

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Met Ala Thr Leu Val Ser Phe Thr Lys Gln Tyr Gln Val Gln Lys Thr

1 5 10 15

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

20 25 30

Ala Lys Gly Phe Ile Asn Asp Asp Leu Lys Arg Asp Glu Asn Tyr Met

35 40 45

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

50 55 60

Thr Leu Val Asn Val Asp Tyr Asp Trp Arg Ser Leu Ala Thr Ala Ile

65 70 75 80

Lys Asn Tyr Arg Lys Asp Arg Ser Asp Thr Asn Lys Lys Asn Leu Glu

85 90 95

Lys Thr Gln Glu Ala Ala Arg Lys Glu Ile Ile Ala Trp Phe Glu Gly

100 105 110

Lys Arg Gly Asn Ser Ala Phe Lys Asn Asn Gln Lys Ser Phe Tyr Gly

115 120 125

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

130 135 140

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

145 150 155 160

Phe Thr Thr Tyr Phe Val Gly Phe His Glu Asn Arg Lys Asn Met Tyr

165 170 175

Ser Thr Glu Ala Lys Ser Thr Ser Val Ala Tyr Arg Val Val Asn Glu

180 185 190

Asn Phe Ser Lys Phe Leu Ser Asn Cys Glu Ala Phe Ser Val Leu Glu

195 200 205

Ala Val Cys Pro Asn Val Leu Val Glu Ala Glu Gln Glu Leu His Leu

210 215 220

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

225 230 235 240

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

245 250 255

Ile Ile Gly Gly Ile Ser Ser Ala Glu Gly Val Arg Lys Ile Arg Gly

260 265 270

Val Asn Glu Val Val Asn Asn Ala Ile Gln Gln Asn Asp Glu Leu Lys

275 280 285

Val Ala Leu Arg Asn Lys Gln Phe Thr Met Val Gln Leu Phe Lys Gln

290 295 300

Ile Leu Ser Asp Arg Ser Thr Leu Ser Phe Val Ser Glu Gln Phe Thr

305 310 315 320

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

325 330 335

Val Asn Asn Lys Val Leu Ala Val Val Lys Thr Leu Phe Glu Asn Phe

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Ala Glu Leu Ala Ser Tyr Asn Asp Lys Tyr Leu Asp Lys Glu Gly Asn

420 425 430

Asp Lys Glu Ile Cys Ser Ile Ala Asn Val Val Leu Glu Ala Val Gly

435 440 445

Ala Leu Glu Ile Met Leu Ala Glu Ser Leu Pro Ala Asp Leu Lys Thr

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Pro Tyr Ser Val Glu Lys Phe Lys Leu Asn Phe Ala Met Pro Thr Leu

530 535 540

Ala Asp Gly Trp Asp Lys Asn Lys Glu Arg Asp Asn Gly Ser Ile Ile

545 550 555 560

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

565 570 575

Lys Pro Val Ile Asp Asn Ala Val Cys Asn Asp Ala Lys Gly Tyr Gln

580 585 590

Lys Met Val Tyr Lys Met Phe Pro Glu Ile Ser Lys Met Val Thr Lys

595 600 605

Cys Ser Thr Gln Leu Asn Ala Val Lys Ala His Phe Glu Asp Asn Thr

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Tyr His Lys Ala Leu Glu Thr Trp Ile Asp Phe Val Lys Glu Phe Leu

675 680 685

Ser Lys Tyr Arg Ser Thr Ala Ile Tyr Asp Leu Thr Thr Leu Leu Pro

690 695 700

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

705 710 715 720

Leu Cys Tyr Lys Ile Asp Tyr Glu Asn Ile Ser Val Glu Gln Val Asn

725 730 735

Glu Trp Val Glu Glu Gly Asn Leu Tyr Leu Phe Lys Ile Tyr Asn Lys

740 745 750

Asp Phe Ala Thr Gly Ser Thr Gly Lys Pro Asn Leu His Thr Met Tyr

755 760 765

Trp Asn Ala Val Phe Ala Glu Glu Asn Leu His Asp Val Val Val Lys

770 775 780

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

785 790 795 800

Lys Val Glu His Arg Val Gly Glu Lys Leu Val Asn Arg Lys Asn Val

805 810 815

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

820 825 830

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

835 840 845

Glu Leu Pro Leu Ala Ile Ile Lys Asp Val Lys His Asn Ile Thr Lys

850 855 860

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

865 870 875 880

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

885 890 895

Gln Ala Phe Leu Lys Asn Asn Pro Asp Val Asn Ile Ile Gly Ile Asp

900 905 910

Arg Gly Glu Arg Asn Leu Leu Tyr Val Val Val Ile Asp Gln Gln Gly

915 920 925

Asn Ile Ile Asp Lys Lys Gln Val Ser Tyr Asn Lys Val Asn Gly Tyr

930 935 940

Asp Tyr Tyr Glu Lys Leu Asn Gln Arg Glu Lys Glu Arg Ile Glu Ala

945 950 955 960

Arg Gln Ser Trp Gly Ala Val Gly Lys Ile Lys Glu Leu Lys Glu Gly

965 970 975

Tyr Leu Ser Leu Val Val Arg Glu Ile Ala Asp Met Met Val Lys Tyr

980 985 990

Asn Ala Ile Val Val Met Glu Asn Leu Asn Ala Gly Phe Lys Arg Val

995 1000 1005

Arg Gly Gly Ile Ala Glu Lys Ala Val Tyr Gln Lys Phe Glu Lys

1010 1015 1020

Met Leu Ile Asp Lys Leu Asn Tyr Leu Val Phe Lys Asp Val Glu

1025 1030 1035

Ala Lys Glu Ala Gly Gly Val Leu Asn Ala Tyr Gln Leu Thr Asp

1040 1045 1050

Lys Phe Asp Ser Phe Glu Lys Met Gly Asn Gln Ser Gly Phe Leu

1055 1060 1065

Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys Ile Asp Pro Val Thr

1070 1075 1080

Gly Phe Ala Asn Val Phe Ser Thr Lys His Ile Thr Asn Thr Glu

1085 1090 1095

Ala Lys Lys Glu Phe Ile Cys Ser Phe Asn Ser Leu Arg Tyr Asp

1100 1105 1110

Glu Ala Lys Asp Lys Phe Val Leu Glu Cys Asp Leu Asn Lys Phe

1115 1120 1125

Lys Ile Val Ala Asn Ser His Ile Lys Asn Trp Lys Phe Ile Ile

1130 1135 1140

Gly Gly Lys Arg Ile Val Tyr Asn Ser Lys Asn Lys Thr Tyr Met

1145 1150 1155

Glu Lys Tyr Pro Cys Glu Asp Leu Lys Ala Thr Leu Asn Ala Ser

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

Trp Ala Ile Met Asn Thr Leu Gln Met Arg Asn Ser Asn Ala Leu

1205 1210 1215

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

1220 1225 1230

Lys Val Phe Asp Ser Arg Thr Cys Gly Ala Glu Leu Pro Lys Asp

1235 1240 1245

Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Leu Tyr

1250 1255 1260

Leu Leu Gln Arg Ile Asp Ile Ser Glu Glu Gly Glu Lys Val Asp

1265 1270 1275

Leu Ser Ile Lys Asn Glu Glu Trp Phe Lys Phe Val Gln Gln Lys

1280 1285 1290

Glu Tyr Ala Arg

1295

<210> 3

<211> 1288

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas-sf3

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Met Tyr Ser Leu Ile Asn Tyr Phe Thr Thr Phe Thr Gly Asn Phe Ile

1 5 10 15

Asn Asn Leu Phe Thr Leu Thr Glu Tyr Ile Met Lys Thr Phe Gln Gln

20 25 30

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

35 40 45

Pro Ile Gly Ser Thr Leu Glu His Ile Asn Lys Asn Gly Leu Leu Asp

50 55 60

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

65 70 75 80

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

85 90 95

Leu Gln Tyr Asp Asn Glu Gly Arg Asn Asn Ser Ile Ser Glu Phe Tyr

100 105 110

Thr Cys Tyr Met Ile Lys Ser Lys Asp Asp Asn Gln Arg Lys Leu Tyr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Gln Asp Ile Gln Ile Ile Glu Glu Phe Lys Asn Phe Thr Thr Tyr Phe

180 185 190

Thr Gly Phe His Glu Asn Arg Lys Asn Met Tyr Thr Ser Glu Ala Gln

195 200 205

Ser Thr Ala Ile Ala Tyr Arg Leu Ile His Glu Asn Leu Pro Lys Phe

210 215 220

Ile Asp Asn Ile Met Val Phe Asp Lys Val Ala Ala Ser Pro Ile Ala

225 230 235 240

Asp Ser Phe Ser Glu Leu Tyr Thr Asn Phe Glu Glu Cys Leu Asn Val

245 250 255

Met Ser Ile Glu Glu Met Phe Lys Leu Asn Tyr Phe Asn Val Val Leu

260 265 270

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

275 280 285

Ile Asp Asn Thr Asn Ile Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn

290 295 300

Leu Tyr Asn Gln Gln Gln Lys Asp Lys Ser Ala Arg Leu Pro Lys Leu

305 310 315 320

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

325 330 335

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

340 345 350

Lys Ala Tyr Gln Glu Leu Asp Glu Gln Val Leu Asn Arg Lys Ile Glu

355 360 365

Gly Glu His Ser Leu Arg Glu Leu Leu Val Gly Leu Ala Asp Tyr Asp

370 375 380

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

385 390 395 400

Gln Lys Val Phe Gly His Trp Gly Val Ile Ser Lys Ala Leu Leu Glu

405 410 415

Glu Leu Lys Asn Glu Val Pro Lys Lys Ser Lys Lys Glu Ser Asp Glu

420 425 430

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

435 440 445

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

450 455 460

Gln Lys Thr Ile Gln Gly Tyr Phe Ala Glu Leu Gly Ala Val Asn Asn

465 470 475 480

Glu Thr Ile Gln Lys Glu Asn Leu Phe Ala Gln Ile Glu Asn Ala Tyr

485 490 495

Thr Glu Val Lys Asp Leu Leu Asn Thr Pro Tyr Thr Gly Lys Asn Leu

500 505 510

Ala Gln Asp Lys Val Asn Val Glu Lys Ile Lys Asn Leu Leu Asp Ala

515 520 525

Ile Lys Ala Leu Gln His Phe Ile Lys Pro Leu Leu Gly Asp Gly Thr

530 535 540

Glu Pro Glu Lys Asp Glu Lys Phe Tyr Gly Glu Phe Ala Ala Leu Trp

545 550 555 560

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

565 570 575

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

580 585 590

Asn Ser Thr Leu Met Asp Gly Trp Asp Leu Asn Lys Glu Gln Ala Asn

595 600 605

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

610 615 620

Asn Lys Lys His Asn Arg Val Phe Asp Val Lys Ala Met Pro Asp Asp

625 630 635 640

Gly Asp Cys Tyr Glu Lys Met Glu Tyr Lys Leu Leu Pro Gly Ala Asn

645 650 655

Lys Met Leu Pro Lys Val Phe Phe Ser Lys Ser Arg Ile Gln Glu Phe

660 665 670

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

675 680 685

Lys Gly Val Thr Phe Asn Ile Lys Asp Cys His Ala Leu Ile Asp Phe

690 695 700

Phe Lys Ala Ser Ile Asn Lys His Glu Asp Trp Cys Lys Phe Gly Phe

705 710 715 720

Arg Phe Ser Pro Thr Glu Thr Tyr Glu Asp Leu Ser Gly Phe Tyr Arg

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

His Thr Leu Tyr Trp Lys Met Leu Phe Asp Glu Lys Asn Leu Ala Asp

785 790 795 800

Val Val Tyr Lys Leu Asn Gly Gln Ala Glu Val Phe Phe Arg Lys Ser

805 810 815

Ser Ile Asn Tyr Glu Gln Pro Thr His Pro Ala Asn Lys Ala Ile Asp

820 825 830

Asn Lys Asn Glu Leu Asn Lys Lys Lys Gln Ser Leu Phe Thr Tyr Asp

835 840 845

Leu Ile Lys Asp Lys Arg Tyr Thr Ile Asp Lys Phe Gln Phe His Val

850 855 860

Pro Ile Thr Met Asn Phe Lys Ser Thr Gly Asn Asp Asn Ile Asn Gln

865 870 875 880

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

885 890 895

Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Leu Thr Val Ile Asn Leu

900 905 910

Lys Gly Glu Ile Lys Glu Gln Tyr Ser Leu Asn Glu Ile Val Asn Thr

915 920 925

Tyr Lys Gly Asn Glu Tyr Arg Thr Asp Tyr His Asp Leu Leu Ser Lys

930 935 940

Arg Glu Asp Glu Arg Met Lys Ala Arg Gln Ser Trp Gln Thr Ile Glu

945 950 955 960

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

965 970 975

Ile Ala Glu Leu Met Ile Lys Tyr Asn Ala Ile Val Val Leu Glu Asp

980 985 990

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

995 1000 1005

Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Leu

1010 1015 1020

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

1025 1030 1035

Ala Tyr Gln Leu Thr Asn Lys Phe Val Ser Phe Gln Lys Met Gly

1040 1045 1050

Lys Gln Cys Gly Phe Leu Phe Tyr Thr Gln Ala Trp Asn Thr Ser

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

Ser Ile Arg Tyr Asn Asp Glu Lys Asp Trp Phe Glu Phe Ala Phe

1100 1105 1110

Asp Tyr Thr Asn Phe Thr Ser Lys Ala Asp Gly Ser Arg Thr Asn

1115 1120 1125

Trp Lys Leu Cys Thr Tyr Tyr Gly Lys Arg Ile Glu Thr Phe Arg Asp

1130 1135 1140

Glu Lys Gln Asn Ser Asn Trp Thr Ser Lys Glu Val Val Leu Thr

1145 1150 1155

Asp Lys Phe Lys Glu Phe Phe Lys Glu Ser Asn Ile Asp Ile His

1160 1165 1170

Ser Asn Leu Lys Glu Ala Ile Met Gln Gln Asp Ser Ala Asp Phe

1175 1180 1185

Phe Lys Lys Leu Leu Tyr Leu Leu Lys Leu Thr Leu Gln Met Arg

1190 1195 1200

Asn Ser Glu Thr Gly Thr Asn Val Asp Tyr Met Gln Ser Pro Val

1205 1210 1215

Ala Asp Glu Glu Gly Asn Phe Tyr Asn Ser Asp Thr Cys Asp Ser

1220 1225 1230

Ser Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1235 1240 1245

Arg Lys Gly Leu Trp Ile Val Gln Gln Ile Lys Thr Ser Asp Asp

1250 1255 1260

Leu Arg Asn Leu Lys Leu Ala Ile Thr Asn Lys Glu Trp Leu Gln

1265 1270 1275

Phe Ala Gln Arg Lys Pro Tyr Leu Asp Glu

1280 1285

<210> 4

<211> 1283

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas-sf6

<400> 4

Met Ser Asn Met Gln Gln Tyr Asp Asn Phe Ile Asn His Tyr Ala Ile

1 5 10 15

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

20 25 30

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

35 40 45

Lys Tyr Gln Ile Val Lys Lys Ile Ile Asp Arg Phe His Arg Lys His

50 55 60

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

65 70 75 80

Leu Lys Arg Phe Glu Glu Leu Tyr Trp Lys Lys Asn Lys Asn Glu Asn

85 90 95

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

100 105 110

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

115 120 125

Val Gln Gln Arg Tyr Gly Ile Leu Phe Asp Ala Lys Ile Phe Lys Asp

130 135 140

Lys Glu Phe Ile Ser Thr Ala Cys Asp Asp Ile Glu Lys Asp Ala Ile

145 150 155 160

Glu Ala Phe Lys Arg Phe Ala Thr Tyr Phe Thr Gly Phe His Glu Asn

165 170 175

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

180 185 190

Arg Val Ile Asn Glu Asn Leu Pro Arg Phe Leu Glu Asn Lys Ala Arg

195 200 205

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

210 215 220

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

225 230 235 240

Phe Thr Leu Asn Tyr Phe Gln Asn Thr Leu Ser Gln Ala Gly Ile Thr

245 250 255

Tyr Tyr Asn Thr Ile Leu Gly Gly Lys Thr Lys Glu Asn Gly Glu Lys

260 265 270

Val Gln Gly Leu Asn Glu Ile Ile Asn Leu Phe Asn Gln Lys Asn Lys

275 280 285

Asp Thr Met Leu Pro Leu Leu Lys Pro Leu Tyr Lys Gln Ile Leu Ser

290 295 300

Glu Glu Tyr Ser Thr Ser Phe Thr Ile Ser Ala Phe Glu Lys Asp Asn

305 310 315 320

Asp Val Leu Gln Ala Ile Gly Ser Phe Cys Asn Asp Cys Ile Phe Tyr

325 330 335

Ala Lys Asn Asn Val Asn Gly Lys Ala Tyr Asn Leu Leu Gln Thr Val

340 345 350

Gln Ala Phe Cys Asn Ser Ile Asp Thr Tyr Asn Asp Asn Arg Leu Asp

355 360 365

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

370 375 380

Tyr Gly Glu Trp Asn Ile Leu Arg Asp Ala Leu Gln Ile His Tyr Glu

385 390 395 400

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

405 410 415

Lys Thr Phe Ser Trp Lys Ala Leu Lys Asp Ala Leu Thr Thr Tyr Lys

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Thr Ile Leu Gln Glu Asp Arg Asn Asn Lys Asn Asn Leu His Gln Glu

485 490 495

Lys Glu Lys Val Ala Thr Ile Lys Gly Phe Leu Asp Ser Val Lys Tyr

500 505 510

Leu Gln Trp Phe Leu Asn Leu Met Tyr Ile Ala Ser Pro Val Asp Asp

515 520 525

Lys Asp Tyr Asp Phe Tyr Asn Glu Leu Glu Met Tyr His Asp Thr Leu

530 535 540

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

545 550 555 560

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

565 570 575

Leu Leu Asp Gly Trp Asp Lys Asn Lys Glu Arg Ala Asn Leu Gly Val

580 585 590

Ile Leu Arg Lys Gly Asn Asn Tyr Tyr Leu Gly Ile Met Asn Lys Lys

595 600 605

Tyr Asn Asp Ile Phe Asp Ser Ile Pro Gly Leu Thr Thr Thr Asp Tyr

610 615 620

Cys Glu Lys Met Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu

625 630 635 640

Pro Lys Val Phe Phe Ser Lys Lys Gly Val Gln Phe Tyr Lys Pro Ser

645 650 655

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

660 665 670

Thr Phe Asn Lys Asn Ser Leu His Lys Leu Ile Asn Phe Tyr Lys Glu

675 680 685

Ser Ile Ala Lys Thr Glu Asp Trp Ser Val Phe Gln Phe Lys Phe Lys

690 695 700

Asn Thr Asn Asp Tyr Ala Asp Ile Ser Gln Phe Tyr Lys Asp Val Glu

705 710 715 720

Arg Gln Gly Tyr Lys Ile Ser Phe Asp Lys Ile Asp Trp Glu Tyr Ile

725 730 735

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

740 745 750

Lys Asp Phe Ser Pro Tyr Ser Lys Gly Lys Pro Asn Leu His Thr Ile

755 760 765

Tyr Trp Lys Asn Ile Phe Ser His Asp Asn Leu Asn Asn Val Val Tyr

770 775 780

Lys Leu Asn Gly Glu Ala Glu Val Phe Tyr Arg Lys Lys Ser Ile Glu

785 790 795 800

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

805 810 815

Asp Lys Phe Lys Tyr Pro Ile Ile Lys Asp Lys Arg Tyr Ala Glu Asp

820 825 830

Lys Phe Leu Phe His Val Pro Ile Thr Met Asn Phe Leu Ser Lys Gly

835 840 845

Glu Pro Asn Ile Asn Gln Arg Val Gln Gln Tyr Ile Ala Ser Thr Ser

850 855 860

Glu Asp Tyr His Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu

865 870 875 880

Tyr Leu Ser Leu Ile Asp Ala Thr Gly Lys Ile Ile Lys Gln Leu Ser

885 890 895

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

900 905 910

Ala Lys Leu Asp Glu Lys Glu Lys Lys Arg Glu Glu Ala Arg Lys Asn

915 920 925

Trp Asp Val Ile Glu Asn Ile Lys Glu Leu Lys Glu Gly Tyr Leu Ser

930 935 940

Gln Val Val His Gln Ile Ala Lys Leu Met Val Glu Tyr Lys Ala Ile

945 950 955 960

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

965 970 975

Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Met Ile Asp Lys

980 985 990

Leu Asn Tyr Leu Val Leu Lys Asp Arg Gln Ala Thr Gln Pro Gly Gly

995 1000 1005

Ser Leu Lys Ala Tyr Gln Leu Ala Ser Ser Leu Glu Ser Phe Lys

1010 1015 1020

Lys Leu Gly Lys Gln Cys Gly Met Ile Phe Tyr Val Pro Ala Val

1025 1030 1035

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

1040 1045 1050

Arg Val Asp Val Ser Thr Leu Asn Ser Ala His Ser Phe Phe Asn

1055 1060 1065

Arg Phe Asn Ala Ile Val Tyr Asn Asn Glu Gln Asp Tyr Phe Glu

1070 1075 1080

Phe His Cys Thr Tyr Lys Asn Phe Val Ser Glu Pro Ser Leu Gln

1085 1090 1095

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

1100 1105 1110

Asp Thr Thr Trp Val Leu Cys Ser Thr His His Glu Arg Tyr Lys

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

Asn Thr Lys Leu Leu Lys Gly Leu Gly Glu Gln Leu Asn Ile Leu

1175 1180 1185

Leu Ala Met Arg Tyr Asn Asn Gly Lys His Gly Asn Glu Glu Lys

1190 1195 1200

Asp Tyr Ile Val Ser Pro Val Lys Asn Asn Tyr Gly Lys Phe Phe

1205 1210 1215

Cys Thr Leu Asp Gly Asp Ala Ser Leu Pro Val Asp Ala Asp Ala

1220 1225 1230

Asn Gly Ala Tyr Ala Ile Ala Leu Lys Gly Leu Met Leu Val Glu

1235 1240 1245

Arg Met Lys Ser Asn Lys Asp Ile Lys Gly Arg Ile Asp Tyr Phe

1250 1255 1260

Ile Ser Asn Asn Glu Trp Phe Asn Tyr Leu Ile Ala Lys Asn Thr

1265 1270 1275

Leu Asn Lys Ser Lys

1280

<210> 5

<211> 1275

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas-sf8

<400> 5

Met Arg Lys Ser Phe Lys Asp Phe Thr Asn Met Tyr Pro Val Gln Lys

1 5 10 15

Thr Leu Arg Phe Glu Leu Lys Pro Leu Gly Lys Thr Glu Gln His Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Glu Tyr Asp Gly Glu Asp Lys Asp Lys Arg Leu Glu Thr Val Gln

85 90 95

Lys Asp Lys Arg Ser Lys Ile Ile Cys Arg Phe Thr Glu Gln Pro Glu

100 105 110

Phe Lys Lys Leu Phe Gly Lys Glu Leu Phe Ser Glu Leu Leu Pro Glu

115 120 125

Met Ile Asn Ala Glu Asn Ala Asp Asn Lys Asp Glu Lys Leu His Ala

130 135 140

Ala Ala Ala Phe Asp Lys Phe Ser Thr Tyr Phe Lys Gly Phe His Asp

145 150 155 160

Asn Arg Arg Asn Ile Tyr Ser Asn Glu Glu Ile Ser Thr Ser Val Ala

165 170 175

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

180 185 190

Thr Phe Lys Thr Ile Cys Lys Lys Ala Pro Glu Ile Ile Glu Gln Thr

195 200 205

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

210 215 220

Phe Arg Ile Glu Ser Phe Asn Asn Val Met Thr Gln Asp Gly Ile Asp

225 230 235 240

Tyr Tyr Asn Asn Ile Ile Asp Gly Val Pro Cys Glu Ala Gly Lys Lys

245 250 255

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

260 265 270

Pro Asp Thr Lys Ile Gln Ile Lys Met Val Pro Leu Tyr Lys Gln Ile

275 280 285

Leu Ser Asp Arg Ala Thr Leu Ser Phe Met Pro Ala Ala Leu Asp Asn

290 295 300

Asp Gly Asp Ala Phe Glu Ala Val Ala Gly Leu Glu Lys Met Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

Gln Lys Ser Val Pro Val Val Ser Ala Ala Leu Phe Gly Ser Trp Asp

355 360 365

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

370 375 380

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

385 390 395 400

Phe Ser Phe Ala Ser Leu Asp Ala Ala Ala Asp Phe Tyr Lys Asp Ser

405 410 415

Leu Pro Gly Glu Lys Ser Ala Arg Arg Ile Lys Asp Tyr Phe Ala Gly

420 425 430

Cys Arg Glu Leu Val Lys Asn Thr Ser Glu Lys Gln Lys Glu Phe Asp

435 440 445

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

450 455 460

Val Lys Ala Tyr Leu Asp Ser Leu Asn Asp Ile Leu Arg Phe Met Arg

465 470 475 480

Pro Phe Glu Thr Glu Asp Ile Thr Asp Ile Asp Thr Glu Phe Tyr Ser

485 490 495

Ala Tyr Ser Val Leu Leu Glu Lys Ile Lys Met Val Ile Pro Val Tyr

500 505 510

Asn Thr Val Arg Asn Tyr Val Thr Lys Lys Pro Phe Lys Thr Asp Lys

515 520 525

Phe Lys Leu Asn Phe Glu Asn Pro Thr Leu Ala Tyr Gly Trp Asp Lys

530 535 540

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

545 550 555 560

Tyr Tyr Leu Gly Ile Met Asn Ala Lys His Lys Ile Lys Pro Ala Glu

565 570 575

Leu Ala Asp Asp His Asn Gly Asp Gly Tyr Lys Lys Met Gln Tyr Met

580 585 590

Gln Met Ser Gly Pro Thr Lys Asp Leu Pro Asn Leu Leu Val Ile Asp

595 600 605

Gly Lys Thr Val Arg Lys Thr Gly Ser Lys Asp Ala Asn Gly Val Asn

610 615 620

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

625 630 635 640

Glu Ile Arg Leu Asp Gly Ser Tyr Leu Glu Thr Ser Asn Asn Phe Ser

645 650 655

Lys Lys Asn Ser Gln Lys Tyr Leu Ala Tyr Tyr Met Lys Leu Leu Lys

660 665 670

Glu Tyr Lys Ser Asn Phe Asp Phe Asn Phe Lys Lys Ala Asn Glu Tyr

675 680 685

Glu Ser Tyr Tyr Asp Phe Thr Asn Asp Ile Lys Lys Gln Cys Tyr Ser

690 695 700

Leu Thr Phe Thr Asn Leu Ala Glu Asn Lys Val Asp Lys Trp Val Asp

705 710 715 720

Glu Gly Arg Leu Tyr Leu Phe Gln Ile Trp Asn Lys Asp Phe Ala Glu

725 730 735

Gly Val Ser Gly Arg Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu

740 745 750

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

755 760 765

Ala Glu Leu Phe Phe Arg Arg Lys Ser Ile Asn Glu Pro Val Val His

770 775 780

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

785 790 795 800

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

805 810 815

Arg Ala Leu Gly Ser Leu Gly Ala Ala Ala Arg Ala Leu Val Glu Ser

820 825 830

Lys Arg Val Arg Ile Thr Asp Val Lys His Glu Leu Val Lys Asp Lys

835 840 845

Arg Tyr Thr Gln Asp Lys Phe Phe Phe His Val Ser Leu Thr Ile Asn

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

Glu Gln Lys His Phe Asn Thr Val Gly Gly Met Asp Tyr His Ala Lys

915 920 925

Leu Asp Gln Arg Glu Lys Ala Arg Asp Glu Ala Arg Lys Ser Trp Lys

930 935 940

Thr Ile Gly Asn Ile Lys Glu Leu Lys Glu Gly Tyr Leu Ser Gln Val

945 950 955 960

Ile His Glu Ile Thr Lys Met Ala Val Glu Asn Asp Ala Ile Ile Ala

965 970 975

Met Glu Asp Leu Asn Val Gly Phe Lys Arg Gly Arg Phe Lys Val Glu

980 985 990

Lys Gln Val Tyr Gln Lys Phe Glu Glu Met Leu Ile Asn Lys Leu Asn

995 1000 1005

Tyr Leu Ser Phe Lys Asp Thr Gly Glu Asn Lys Gln Cys Gly Ile

1010 1015 1020

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

1025 1030 1035

Ile Gly Lys Gln Cys Gly Ile Ile Phe Tyr Val Pro Ala Gly Tyr

1040 1045 1050

Thr Ser Lys Ile Asp Pro Val Thr Gly Phe Val Ser Val Phe Asn

1055 1060 1065

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

1070 1075 1080

Arg Leu Asp Ser Ile Arg Tyr Asp Lys Lys Leu Asp Met Phe Val

1085 1090 1095

Phe Ser Phe Asp Tyr Ser Glu Phe Lys Thr Tyr Gln Thr Leu Pro

1100 1105 1110

Val Thr Lys Trp Asp Val Tyr Thr Asn Gly Lys Arg Ile Ile Asn

1115 1120 1125

Lys Arg Glu Gly Ser Arg Trp Ile Pro Gln Asn Val Val Pro Thr

1130 1135 1140

Glu Glu Met Lys Arg Thr Leu Lys Gln Leu Gly Ile Glu Tyr Glu

1145 1150 1155

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

1160 1165 1170

Leu Ala Ser Asp Val Phe Tyr Ile Phe Lys Asn Thr Leu Gln Met

1175 1180 1185

Arg Asn Ser Asn Ala Ala Thr Gly Glu Asp Tyr Ile Ile Ser Pro

1190 1195 1200

Val Lys Gly Lys Lys Gly Val Phe Phe Ser Ser Ser Ala Lys Asp

1205 1210 1215

Lys Ser Leu Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile

1220 1225 1230

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

1235 1240 1245

Lys Asp Asp Gly Lys Met Ser Tyr Lys Asp Met Tyr Ile Ser Asn

1250 1255 1260

Pro Asp Trp Phe Lys Phe Met Gln Thr Gly Lys His

1265 1270 1275

<210> 6

<211> 1273

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas-sf9

<400> 6

Met Lys Glu Asn Phe Ile Gly Lys Tyr Gln Ile Thr Lys Thr Leu Arg

1 5 10 15

Phe Ser Leu Ile Pro Ile Gly Lys Thr Glu Glu Tyr Phe Asn Ala Arg

20 25 30

Cys Met Leu Glu Glu Asp Glu Gln Arg Ala Glu Asp Tyr Val Lys Val

35 40 45

Lys Ser Phe Ile Asp Glu Tyr His Lys Ala Phe Ile Glu Arg Ile Leu

50 55 60

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

65 70 75 80

Glu Lys Val Arg Asp Tyr Ala Asp Leu Tyr Asn Ser Ser Gln Arg Asp

85 90 95

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

100 105 110

Glu Ala Phe Thr Lys Asp Asp His Tyr Asp Arg Leu Phe Asn Lys Asp

115 120 125

Ile Ile Glu Glu Leu Leu Pro Glu Tyr Leu Gly Asp Ser Arg Lys Glu

130 135 140

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

145 150 155 160

Gly Phe Phe Glu Asn Arg Lys Asn Met Tyr Val Lys Glu Gln Glu Thr

165 170 175

Thr Ala Ile Ala Tyr Arg Cys Ile Asp Glu Asn Leu Pro Arg Phe Leu

180 185 190

Asp Asn Ala Thr Ile Trp Lys Lys Lys Leu Arg Asp Ala Leu Pro Glu

195 200 205

Glu Asp Ile Cys Arg Leu Asn Lys Glu Cys Thr Asp Phe His Asp Lys

210 215 220

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

225 230 235 240

Gln Ser Gly Ile Asp Trp Tyr Asn Gln Ile Leu Gly Gly Tyr Thr Lys

245 250 255

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

260 265 270

Asn Asp Lys Val Ser Glu Lys Glu Arg Ser His Arg Leu Pro Leu Leu

275 280 285

Lys Pro Leu Tyr Lys Gln Ile Leu Ser Asp Arg Val Ser Thr Ser Phe

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Tyr His Glu Asn Asn Pro Leu Gly Lys Arg Glu Ser Ile Glu Leu Tyr

385 390 395 400

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

405 410 415

Glu Glu Ile Gln Gln Phe Gly Glu Gly Lys Ala Lys Glu Glu Tyr Arg

420 425 430

Asn Glu Thr Val Ile His Phe Tyr Ser Gly Thr Val Arg Lys Gln Ser

435 440 445

Asn Lys Ile Cys Asp Ser Tyr Lys Asp Ala Tyr Lys Arg Ile Lys Pro

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Asp Asn Asn Phe Tyr Asn Arg Phe Leu Val Ala Phe Asp Thr Phe Thr

515 520 525

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

530 535 540

Pro Phe Ser Thr Glu Lys Ile Lys Leu Asn Phe Asn Asn Pro Gln Phe

545 550 555 560

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

565 570 575

Leu Arg Ser Ala Gly Lys Tyr Tyr Leu Gly Val Met Asp Thr Lys Ser

580 585 590

Lys His Ser Phe Lys Lys Tyr Pro Ser Pro Lys Ser Lys Asn Asp Val

595 600 605

Val Glu Lys Met Phe Leu His Gln Val Ala Asn Pro Ala Lys Asp Val

610 615 620

Gln Asn Leu Met Val Ile Asn Gly Lys Thr Val Arg Arg Thr Gly Arg

625 630 635 640

Lys Glu Thr Glu Gly Glu Tyr Lys Gly Glu Asn Leu Arg Leu Glu Glu

645 650 655

Leu Lys Asn Thr His Leu Pro Glu Glu Ile Asn Arg Ile Arg Lys Ser

660 665 670

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

675 680 685

Val Ala Phe Ile Lys Phe Tyr Met Glu Arg Thr Lys Glu Tyr Tyr Thr

690 695 700

Asn Ser His Phe Glu Phe Arg Asn Ala Glu Asn Tyr Gln Asp Phe Lys

705 710 715 720

Glu Phe Thr Asp Asp Ile Asp Ala Gln Ala Tyr Gln Val His Phe Lys

725 730 735

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

740 745 750

Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Pro Tyr Ser Arg Gly

755 760 765

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

770 775 780

Asn Leu Ala Asp Val Val Phe Lys Leu Asp Gly Asn Ala Glu Met Phe

785 790 795 800

Tyr Arg Lys Ala Ser Leu Lys Lys Gln Ile Thr His Pro Ala Asn Lys

805 810 815

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

820 825 830

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

835 840 845

Leu His Phe Pro Ile Thr Leu Asn Phe Lys Glu Ala Lys Asn Ala Asn

850 855 860

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

865 870 875 880

Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Cys Val

885 890 895

Ile Asp Gly Asn Gly Asn Ile Val Glu Gln Met Ser Met Asn Glu Ile

900 905 910

Thr Thr Asp Asn Asn Tyr Lys Val Asn Tyr His Asn Leu Leu Gln Arg

915 920 925

Lys Glu Glu Glu Arg Lys Lys Ala Arg Gly Asn Trp Ser Val Ile Glu

930 935 940

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

945 950 955 960

Ile Cys Gly Leu Val Ile Lys Tyr Asn Ala Val Ile Ala Met Glu Asn

965 970 975

Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Arg Val Glu Lys Gln Val

980 985 990

Tyr Gln Lys Phe Glu Asn Asn Leu Ile Lys Lys Leu Asn Tyr Leu Ala

995 1000 1005

Asp Lys Lys Leu Pro Pro Glu Gln Asp Gly Gly Leu Leu Arg Ala

1010 1015 1020

Tyr Gln Leu Thr Glu Lys Phe Glu Lys Ile Asn Lys Ser Asn Gln

1025 1030 1035

Asn Gly Ile Ile Phe Phe Val Pro Ala Trp Leu Thr Ser Lys Ile

1040 1045 1050

Asp Pro Thr Thr Gly Phe Thr Asn Leu Leu Tyr Pro Arg Tyr Glu

1055 1060 1065

Ser Val Lys Lys Ala Lys Asn Phe Phe Ala Asn Phe Asn Leu Ile

1070 1075 1080

Thr Tyr Asp Ala Ser Glu Asp Met Phe Arg Phe Asp Phe Asp Tyr

1085 1090 1095

Thr Lys Phe Leu Cys Gly Val Ala Asp Phe Lys Lys Lys Trp Ser

1100 1105 1110

Val Trp Ser Tyr Gly Glu Arg Ile Lys Thr Arg Arg Lys Glu Lys

1115 1120 1125

His Asn Asn Asp Ile Glu Tyr Thr Thr Val Gln Leu Thr Asp Glu

1130 1135 1140

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

1145 1150 1155

Leu Gln Lys Gln Ile Ile Glu Ala Asp Asp Lys Glu Phe Phe Tyr

1160 1165 1170

Ser Leu Tyr Ser Leu Leu Asn Leu Thr Leu Gln Met Arg Asn Ser

1175 1180 1185

Asn Pro Asn Ser Gly Asp Asp Tyr Leu Ile Ser Pro Val Arg Asn

1190 1195 1200

Thr Ser Gly Gly Phe Tyr Asp Ser Arg Asn Tyr Leu Lys Ser Gly

1205 1210 1215

Asn Leu Ser Leu Pro Val Asp Ala Asp Ala Asn Gly Ala Tyr Asn

1220 1225 1230

Ile Ala Arg Lys Cys Leu Trp Gln Ile Met Lys Leu Lys Ser Leu

1235 1240 1245

Ser Glu Asp Glu Thr Lys Lys Pro Asn Leu Thr Ile Ser Asn Lys

1250 1255 1260

Asp Trp Leu Cys Tyr Ala Gln Glu Asn Lys

1265 1270

<210> 7

<211> 1273

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas-sf10

<400> 7

Met Gln Asp Lys Thr Gly Trp Ser Ser Phe Thr Asn Lys Tyr Ser Leu

1 5 10 15

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

20 25 30

Met Leu Glu Asp Asp Gly Val Phe Gln Lys Asp Arg Glu Arg Gln Glu

35 40 45

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

50 55 60

Ile Lys Glu Ala Leu Asn Asn Leu Lys Leu Glu Gly Leu Thr Glu Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Glu Thr Ala Gln Ile Trp Val Glu Lys Tyr Lys Glu Phe Gly Phe Lys

115 120 125

Lys Lys Asn Ile Gly Phe Leu Phe Glu Glu Gly Val Phe Glu Leu Met

130 135 140

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

145 150 155 160

Glu Val Leu Ser Ile Phe Asp Gly Trp Lys Gly Phe Leu Gly Tyr Phe

165 170 175

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

180 185 190

Ser Thr Ala Val Ser Thr Arg Ile Ile Asn Glu Asn Leu Lys Ile Tyr

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Tyr Tyr Asn Glu Ile Leu Gly Gly Lys Thr Leu Lys Asn Gly Thr Lys

260 265 270

Leu Lys Gly Leu Asn Glu Val Ile Asn Glu Tyr Lys Gln Lys Asn Lys

275 280 285

Ser Gly Glu Leu Ser Lys Phe Lys Met Leu Lys Lys Gln Ile Leu Gly

290 295 300

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

305 310 315 320

Leu Lys Asp Val Leu Lys Asp Phe Phe Tyr Asn Ala Asp Pro Lys Ile

325 330 335

Thr Leu Phe Lys Thr Leu Leu Glu Asp Phe Phe Ser Asn Thr Glu Lys

340 345 350

Tyr Lys Asp Glu Leu Asp Lys Ile Tyr Phe Asn Thr Val Ala Ile Asn

355 360 365

Gly Ile Leu His Arg Trp Val Asp Asp Ser Gly Val Phe Gln Lys Tyr

370 375 380

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

385 390 395 400

Asp Lys Lys Glu Asp Ser Tyr Lys Phe Pro Asp Phe Ile Ser Phe Glu

405 410 415

His Ile Lys Val Ala Leu Glu Asn Cys Glu Arg Asp Gly Leu Lys Asp

420 425 430

Lys Phe Trp Lys Glu Lys Tyr Tyr Thr Lys Glu Cys Leu Thr Glu Asn

435 440 445

Gly Leu Ala Asn Leu Trp Gln Glu Phe Leu Glu Ile Tyr Lys Cys Glu

450 455 460

Phe Lys Lys Leu Tyr Asp Tyr Lys Thr Asp Asp Asn Asp Cys Tyr Leu

465 470 475 480

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

485 490 495

Pro Lys Glu Lys Ser Ala Lys Asp Ile Ile Lys Asp Tyr Leu Asp Ser

500 505 510

Val Leu Ser Ile Tyr Gln Leu Ala Lys Tyr Phe Ala Leu Glu Lys Lys

515 520 525

Lys Val Trp Thr Thr Asp Tyr Glu Thr Gly Asp Phe Tyr Tyr Glu Tyr

530 535 540

Ile Lys Phe Tyr Glu Asp Thr Tyr Glu Gln Ile Ile Lys Pro Tyr Asn

545 550 555 560

Leu Val Arg Asn Tyr Leu Thr Arg Lys Pro Ile Asn Thr Ala Lys Lys

565 570 575

Trp Lys Leu Asn Phe Asp Asn Ala Tyr Leu Ala Ser Gly Trp Asp Lys

580 585 590

Asp Lys Glu Val Ser Asn Leu Thr Val Ile Leu Arg Arg Asp Glu Gln

595 600 605

Tyr Tyr Leu Ala Ile Met Lys Lys Gly Lys Asn Lys Ile Phe Glu Lys

610 615 620

Lys Phe Ser Cys Gly Glu Phe Glu Lys Met Glu Tyr Lys Gln Ile Ala

625 630 635 640

Glu Ala Ser Ser Asp Ile His Asn Leu Val Leu Met Asn Asp Gly Ser

645 650 655

Cys Arg Arg Cys Ile Lys Met His Asp Lys Arg Lys Tyr Trp Pro Leu

660 665 670

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

675 680 685

Val Arg Arg Asp Phe Glu Arg Phe Val Asn Tyr Met Lys Lys Cys Ser

690 695 700

Leu Leu Tyr Trp Lys Glu Tyr Asp Leu Lys Phe Ser Asp Thr Ser Thr

705 710 715 720

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

725 730 735

Lys Leu Ser Phe Ser Ala Ile Pro Glu Ser Tyr Ile Asn Glu Lys Asn

740 745 750

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

755 760 765

Ile Lys Thr Glu Gly Asn Lys Asn Leu His Thr Met Tyr Trp Glu Ser

770 775 780

Ile Phe Ser Glu Glu Asn Arg Phe Arg Asn Phe Ile Val Lys Leu Asn

785 790 795 800

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

805 810 815

Glu Gln Arg Asn Phe Thr Arg Glu Ile Ile Lys Asn Arg Arg Tyr Thr

820 825 830

Glu Asn Lys Ile Tyr Phe His Cys Pro Ile Thr Leu Asn Arg Ile Ser

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Glu Asp Ala Thr Gly Ser Phe Asn Thr Ile Gly Ser Thr Asp Tyr His

900 905 910

Arg Leu Leu Glu Glu Lys Ala Lys Asp Arg Glu Lys Glu Arg Lys Asp

915 920 925

Trp Asp Leu Ile Arg Gly Ile Lys Asp Leu Lys Lys Gly Tyr Ile Ser

930 935 940

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

945 950 955 960

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

965 970 975

Met Glu Lys Ser Val Tyr Gln Gln Leu Glu Lys Ala Leu Ile Asn Lys

980 985 990

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

995 1000 1005

His Leu Leu Lys Ala Tyr Gln Leu Ala Ala Pro Phe Gln Thr Phe

1010 1015 1020

Asp Lys Met Gly Arg Gln Thr Gly Ile Ile Phe Tyr Thr Gln Ala

1025 1030 1035

Ser Tyr Thr Ser Lys Ile Asp Pro Ile Thr Gly Trp Arg Pro Asn

1040 1045 1050

Leu Tyr Leu Lys Tyr Arg Asn Ile Asp Asp Ser Lys Glu Ser Ile

1055 1060 1065

Lys Lys Phe Lys Ser Ile Leu Phe Asn Lys Glu Lys Asn Arg Phe

1070 1075 1080

Glu Phe Thr Tyr Asp Leu Lys Asp Phe Val Asp Phe Glu Glu Asp

1085 1090 1095

Lys Ile Pro Glu Lys Thr Glu Trp Thr Leu Cys Ser Ser Val Glu

1100 1105 1110

Arg His Lys Trp Asn Arg His Met Asn Asn Asn Lys Gly Gly Tyr

1115 1120 1125

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

1130 1135 1140

Glu Asn Asn Ile Ser Met Asn Lys Asp Ile Val Asp Gln Val Glu

1145 1150 1155

Ser Ile Ser Asn Gly Asn Phe Phe Arg Gln Phe Ile Tyr Leu Phe

1160 1165 1170

Asn Leu Val Cys Gln Ile Arg Asn Thr Asp Glu Lys Ala Glu Asp

1175 1180 1185

Val Asp Lys Arg Asp Phe Ile Leu Ser Pro Val Glu Pro Phe Phe

1190 1195 1200

Asp Ser Arg Arg Ala Lys Asp Phe Lys Ala Tyr Gly Asp Asn Leu

1205 1210 1215

Pro Lys Asn Gly Asp Glu Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1220 1225 1230

Gly Val Leu Ile Ile Lys Lys Ile Lys Glu Tyr Tyr Asn Gln Asn

1235 1240 1245

Gly Ser Cys Asp Lys Leu Gly Trp Gly Asp Leu Ser Ile Ser His

1250 1255 1260

Lys Glu Trp Asp Asp Phe Ala Thr Asn Asn

1265 1270

<210> 8

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf4

<400> 8

guuuagaagc augcuuuaau uucuacuguu guagau 36

<210> 9

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf1

<400> 9

gucuaaaccu caaugaaaau uucuacuguu guagau 36

<210> 10

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf3

<400> 10

gucuauaaga cuauuauaau uucuacuauu guagau 36

<210> 11

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf6

<400> 11

gucuaaaggu auuauaaaau uucuacuauu guagau 36

<210> 12

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf8

<400> 12

gucuaaaggc cuuauauaau uucuacuuuu guagau 36

<210> 13

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf9

<400> 13

guuuaagacc uccuuuuaau uucuacuguu guagau 36

<210> 14

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf10

<400> 14

cucaauuccu uacaauagau uucuacuuuu guagau 36

<210> 15

<211> 709

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR

<400> 15

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accttcggta 420

taacaacttc gacgagctct acaaagcttg gcgtaatcat ggtcatagct gtttcctgtg 480

tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 540

gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 600

ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 660

ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgc 709

<210> 16

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf4

<400> 16

aauuucuacu guuguagau 19

<210> 17

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf1

<400> 17

aauuucuacu guuguagau 19

<210> 18

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf3

<400> 18

aauuucuacu auuguagau 19

<210> 19

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf6

<400> 19

aauuucuacu auuguagau 19

<210> 20

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf8

<400> 20

aauuucuacu uuuguagau 19

<210> 21

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf9

<400> 21

aauuucuacu guuguagau 19

<210> 22

<211> 19

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas-sf10

<400> 22

gauuucuacu uuuguagau 19

Claims (23)

1. A Cas protein, characterized in that the Cas protein consists of the amino acid sequence shown in SEQ ID No.1. 2. A fusion protein comprising the Cas protein according to claim 1 and other modified parts. 3. An isolated polynucleotide, characterized in that the polynucleotide is a polynucleotide sequence encoding the Cas protein according to claim 1, or a polynucleotide sequence encoding the fusion protein according to claim 2. 4. A vector, characterized in that the vector comprises the polynucleotide according to claim 3 and a regulatory element operably linked thereto. 5. A CRISPR-Cas system, characterized in that the system comprises the Cas protein according to claim 1 and at least one gRNA, wherein the gRNA comprises a direct repeat sequence capable of binding to the Cas protein according to claim 1 and a guide sequence capable of targeting a target sequence. 6. A vector system, characterized in that the vector system comprises one or more vectors, and the one or more vectors comprise: a) a first regulatory element and a gRNA, wherein the first regulatory element is operably connected to the gRNA, and the gRNA comprises a direct repeat sequence capable of binding to the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence; b) a second regulatory element and the Cas protein of claim 1, wherein the second regulatory element is operably connected to the Cas protein of claim 1; Components (a) and (b) are located on the same or different carriers of the system. 7. A composition, characterized in that the composition comprises: (i) a protein component selected from: the Cas protein of claim 1 or the fusion protein of claim 2; (ii) a nucleic acid component selected from: gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a precursor RNA nucleic acid encoding the gRNA; the gRNA comprises a direct repeat sequence capable of binding to the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence; The protein component and the nucleic acid component are combined with each other to form a complex. 8. An engineered host cell, characterized in that the host cell comprises the Cas protein described in claim 1, or the fusion protein described in claim 2, or the polynucleotide described in claim 3, or the vector described in claim 4, or the CRISPR-Cas system described in claim 5, or the vector system described in claim 6, or the composition described in claim 7. 9. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8 in gene editing or gene targeting, wherein the application is for purposes other than disease diagnosis and treatment, and the subject of the application does not include humans. 10. The use according to claim 9, characterized in that the use is in gene cutting. 11. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8 in the preparation of a reagent or kit for gene editing or gene targeting. 12. The use according to claim 11, characterized in that the use is for preparing a reagent or a kit for gene cutting. 13. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8 in any one or more of the following, wherein the application is for purposes other than disease diagnosis and treatment, and the subject of the application does not include humans, and the application is: Non-specific cleavage of side branch nucleic acids; non-specific degradation of side branch nucleic acids; nucleic acid detection. 14 . The use according to claim 13 , characterized in that the non-specific cleavage of side branch nucleic acid is non-specific cleavage of single-stranded nucleic acid. 15. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8 in the preparation of a reagent or kit for use selected from any one or more of the following: non-specific cleavage of side branch nucleic acids; non-specific degradation of side branch nucleic acids; nucleic acid detection. 16 . The use according to claim 15 , characterized in that the non-specific cleavage of side branch nucleic acid is non-specific cleavage of single-stranded nucleic acid. 17. A method for editing a target nucleic acid or targeting a target nucleic acid, characterized in that the method is for non-disease diagnosis and treatment purposes, the subject of application of the method does not include humans, and the method comprises contacting the target nucleic acid with the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8. 18. A method for cutting a target nucleic acid, characterized in that the method is for non-disease diagnosis and treatment purposes, the subject of application of the method does not include humans, and the method comprises contacting the target nucleic acid with the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8. 19. A method for cutting a single-stranded nucleic acid, characterized in that the method is for non-disease diagnosis and treatment purposes, the object of application of the method does not include humans, the method comprises contacting a nucleic acid population with the Cas protein and gRNA according to claim 1, the gRNA comprises a direct repeat sequence capable of binding to the Cas protein according to claim 1 and a guide sequence capable of targeting a target sequence, wherein the nucleic acid population comprises a target nucleic acid and at least one non-target single-stranded nucleic acid, the gRNA is capable of targeting the target nucleic acid, and the Cas protein cuts the non-target single-stranded nucleic acid. 20. A kit for gene editing or gene targeting, characterized in that the kit comprises the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8. 21. A kit for detecting a target nucleic acid in a sample, characterized in that the kit comprises: (a) the Cas protein according to claim 1, or a nucleic acid encoding the Cas protein; (b) gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA comprising the gRNA, or a nucleic acid encoding the precursor RNA; the gRNA comprises a direct repeat sequence capable of binding to the Cas protein according to claim 1 and a guide sequence capable of targeting a target sequence; and (c) a single-stranded nucleic acid detector, which is a single-stranded nucleic acid that does not hybridize with the gRNA. 22. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the host cell of claim 8 in the preparation of a preparation or a kit, wherein the application object of the preparation or the kit does not include humans, and the preparation or the kit is used for: (i) editing a target sequence in a target locus to modify an organism; (ii) Treatment of disease. 23. A method for detecting a target nucleic acid in a sample, characterized in that the method is a method for non-disease diagnosis and treatment purposes, and the method comprises contacting the sample with the Cas protein, gRNA (guide RNA) and a single-stranded nucleic acid detector according to claim 1, wherein the gRNA comprises a region that binds to the Cas protein and a guide sequence that hybridizes with the target nucleic acid; detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector is a single-stranded nucleic acid that does not hybridize with the gRNA.

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