WO2013139994A1 - A novel method of producing an oocyte carrying a modified target sequence in its genome - Google Patents

Abstract

The present invention relates to a method of producing an oocyte carrying a modified target sequence in its genome, the method comprising the steps: (a) introducing into an oocyte a nuclease that specifically binds to and introduces a double-strand break in said target sequence or a nucleic acid molecule encoding said nuclease; and (b) introducing a single stranded oligodesoxynucleotide into the oocyte, wherein the oligodesoxynucleotide comprises a donor nucleic acid sequence and regions homologous to the target sequence; thereby inducing homologous recombination at the target sequence with the donor nucleic acid sequence. The present invention further relates to a method of producing a non-human vertebrate carrying a modified target sequence in its genome, the method comprising: (a) producing an oocyte carrying a modified target sequence in its genome in accordance with the method of the invention; (b) transferring the oocyte obtained in (a) to a pseudopregnant female host; and (c) analysing the offspring delivered by the female host for the presence of the modification.

Description

A novel method of producing an oocyte carrying a modified target sequence in its genome The present invention relates to a method of producing an oocyte carrying a modified target sequence in its genome, the method comprising the steps: (a) introducing into an oocyte a nuclease that specifically binds to and introduces a double-strand break in said target sequence or a nucleic acid molecule encoding said nuclease; and (b) introducing a single stranded oligodesoxynucleotide into the oocyte, wherein the oligodesoxynucleotide comprises a donor nucleic acid sequence and regions homologous to the target sequence; thereby inducing homologous recombination at the target sequence with the donor nucleic acid sequence. The present invention further relates to a method of producing a non-human vertebrate carrying a modified target sequence in its genome, the method comprising: (a) producing an oocyte carrying a modified target sequence in its genome in accordance with the method of the invention; (b) transferring the oocyte obtained in (a) to a pseudopregnant female host; and (c) analysing the offspring delivered by the female host for the presence of the modification.

In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Gene targeting in embryonic stem (ES) cells is routinely applied to modify the mammalian genome, in particular the mouse genome, which established the mouse as the most commonly used genetic animal model (Capecchi MR (2005)). The basis for reverse mouse genetics was initially established in the 1980-ies, when ES cell lines were established from cultured murine blastocysts, culture conditions were identified that maintain their pluripotent differentiation state in vitro (Evans MJ, Kaufman MH., Nature 1981 ; 292:154-6; Martin GR. Proc Natl Acad Sci U S A 1981 ; 78:7634-8) and it was found that ES cells are able to colonize the germ line in chimaeric mice upon microinjection into blastocysts (Bradley et a/., Nature 1984; 309:255-6; Gossler et a/., Proc Natl Acad Sci U S A 1986; 83:9065-9). Since the first demonstration of homologous recombination in ES cells in 1987 (Thomas KR, Capecchi MR., Cell 1987; 51 :503-12) and the establishment of the first knockout mouse strain in 1989 (Schwartzberg PL, Goff SP, Robertson EJ., Science 1989; 246:799-803) gene targeting was adopted to a plurality of genes and has been used in the last decades to generate more than 3000 knockout mouse strains that provided a wealth of information on in vivo gene functions (Collins FS, Rossant J, Wurst W., Cell 2007; 128:9-13; Capecchi, M. R., Nat Rev Genet 2005; 6: 507-12). Accordingly, gene targeting in ES cells has revolutionised the in vivo analysis of mammalian gene function using the mouse as genetic model system. However, at present this reverse genetics approach is restricted to mice, as germ line competent ES cell lines that can be genetically modified could be established only from these animals, so far. The exception from this rule is achieved by homologous recombination in primary cells from pig and sheep followed by the transplantation of nuclei from recombined somatic cells into enucleated oocytes (cloning) (Lai L, Prather RS. 2003. Reprod Biol Endocrinol 2003; 1 :82; Gong M, Rong YS. 2003. Curr Opin Genet Dev 13:215-220). However, since this methodology is inefficient and time consuming it did not develop into a simple routine procedure.

Direct genome editing by zinc-finger nucleases (ZFN) as well as TAL-nucleases in one-cell embryos has been recently established as an alternative mutagenesis approach in mice, rats, rabbits and zebrafish (Carbery et al. (2010); Cui ef a/. (2011 ); Doyon et a/. (2008); Flisikowska ef al. (201 1 ); Mashimo et al. (2010); Meng et al. (2008); Meyer et al. (2010); Geurts AM, et al. (2009); Huang (2011 ; Tesson (2011 )). Such nucleases are designed to induce double-strand breaks (DSBs) at preselected genomic target sites (Klug (2010); Porteus & Carroll (2005); Porteus & Baltimore (2003); Santiago et al. (2008)). DSBs targeted to coding exons frequently undergo sequence deletions leading to gene knockout or allow the insertion (knock-in) of DNA sequences from gene targeting vectors via homologous recombination (HR). The generation of knockout and knock-in mutants at the Rosa26, Mdrla, Pxr, and IgM loci by microinjection of ZFNs one-cell embryos of mice, rats and rabbits (Cui et al. (2011 ); Flisikowska et al. (2011 ); Meyer et al. (2010); Huang (2011 ; Tesson (2011 )) has recently been reported.

Traditional approaches for the generation of precisely targeted mutations, such as the replacement of codons for modelling disease alleles or the insertion of recombinase recognition sites to generate conditional alleles are based on the use of gene targeting vectors which serve as templates for the homologous recombination-mediated transfer of pre-planned sequence modifications into the genome. Typical gene targeting vectors are plasmid-based constructs that include 4 to 10 kb homology regions derived from the target locus to guide the insertion of a desired mutation during the homologous recombination process. However, the construction of gene targeting vectors has the drawback of being a low throughput and time consuming task. In addition, the large size of these DNA constructs limits the number of molecules that can be introduced into embryos without eliciting toxicity. Thus, whereas methods have been described in the art for the targeted modification of endogenous genes, there is still a need to provide improved, e.g. simplified means and methods that expedite the generation of mutant alleles as compared to the time consuming methods presently employed in the art. This need is addressed by the provision of the embodiments characterised in the claims.

Accordingly, the present invention relates to a method of producing an oocyte carrying a modified target sequence in its genome, the method comprising the steps: (a) introducing into an oocyte a nuclease that specifically binds to and introduces a double-strand break in said target sequence or a nucleic acid molecule encoding said nuclease; and (b) introducing a single stranded oligodesoxynucleotide into the oocyte, wherein the oligodesoxynucleotide comprises a donor nucleic acid sequence and regions homologous to the target sequence; thereby inducing homologous recombination at the target sequence with the donor nucleic acid sequence.

In accordance with the present invention, a "modified target sequence" is a nucleotide sequence in which genomic manipulations have led to an alteration of the respective target nucleotide sequence. The term "modified" includes, but is not limited to, one or more nucleotides that are substituted, inserted and deleted within the target sequence.

The term "substitution", as used herein, is defined in accordance with the pertinent art and refers to the replacement of nucleotides with other nucleotides. The term includes for example the replacement of single nucleotides resulting in point mutations. Said point mutations can lead to an amino acid exchange in the resulting protein product but may also not be reflected on the amino acid level. Also encompassed by the term "substitution" are mutations resulting in the replacement of multiple nucleotides, such as for example parts of genes, such as parts of exons or introns as well as the replacement of entire genes. The number of nucleotides that replace the originally present nucleotides may be the same or different (i.e. more or less) as compared to the number of nucleotides removed. Preferably, the number of replacement nucleotides corresponds to the number of originally present nucleotides that are substituted. The term "insertion", in accordance with the present invention, is defined in accordance with the pertinent art and refers to the incorporation of one or more nucleotides into a nucleic acid molecule. Insertion of parts of genes, such as parts of exons or introns as well as insertion of entire genes is also encompassed by the term "insertion". When the number of inserted nucleotides is not dividable by three, the insertion can result in a frameshift mutation within a coding sequence of a gene. Such frameshift mutations will alter the amino acids encoded by a gene following the mutation. In some cases, such a mutation will cause the active translation of the gene to encounter a premature stop codon, resulting in an end to translation and the production of a truncated protein. When the number of inserted nucleotides is instead dividable by three, the resulting insertion is an "in-frame insertion". In this case, the reading frame remains intact after the insertion and translation will most likely run to completion if the inserted nucleotides do not code for a stop codon. However, because of the inserted nucleotides, the finished protein will contain, depending on the size of the insertion, one or multiple new amino acids that may effect the function of the protein.

The term "deletion", as used in accordance with the present invention, is defined in accordance with the pertinent art and refers to the loss of nucleotides or part of genes, such as exons or introns as well as entire genes. As defined with regard to the term "insertion", the deletion of a number of nucleotides that is not evenly dividable by three will lead to a frameshift mutation, causing all of the codons occurring after the deletion to be read incorrectly during translation, potentially producing a severely altered and most likely nonfunctional protein. If a deletion does not result in a frameshift mutation, i.e. because the number of nucleotides deleted is dividable by three, the resulting protein is nonetheless altered as the finished protein will lack, depending on the size of the deletion, one or several amino acids that may effect the function of the protein.

The above defined modifications are not restricted to coding regions in the genome, but can also be introduced into non-coding regions of the target genome, for example in regulatory regions such as promoter or enhancer elements or in introns.

Examples of modifications of the target genome include, without being limiting, the introduction of mutations into a wildtype gene in order to analyse its effect on gene function; the replacement of an entire gene with a mutated gene or, alternatively, if the target sequence comprises mutation(s), the alteration of these mutations to identify which one is causative of a particular effect; the removal of entire genes or proteins or the removal of regulatory elements from genes or proteins as well as the introduction of fusion-partners, such as for example purification tags such as the his-tag or the tap-tag. In accordance with the present invention, the term "target sequence in the genome" refers to the genomic location that is to be modified by the method of the invention. The "target sequence in the genome" comprises but is not restricted to the nucleotide(s) subject to the particular modification and the sequence to which the regions homologous to the target sequence bind. In other words, the term "target sequence in the genome" also comprises the sequence surrounding the relevant nucleotide(s) to be modified. Preferably, the term "target sequence" refers to the entire gene to be modified.

The term "oocyte", as used herein, refers to the female germ cell involved in reproduction, i.e. the ovum or egg cell. In accordance with the present invention, the term "oocyte" comprises both oocytes before fertilisation as well as fertilised oocytes, which are also called zygotes. Thus, the oocyte before fertilisation comprises only maternal chromosomes, whereas an oocyte after fertilisation comprises both maternal and paternal chromosomes. After fertilisation, the oocyte remains in a double-haploid status for several hours, in mice for example for up to 18 hours after fertilisation.

The term "introducing into the oocyte", as used herein, relates to any known method of bringing the nuclease or a nucleic acid molecule encoding the nuclease as well as the single stranded oligodesoxynucleotide into the oocyte. Non-limiting examples include microinjection, infection with viral vectors and electroporation. All these methods are well known in the art and have been described in part in the appended examples.

The terms "nuclease", "endonuclease" and "restriction endonuclease", as used herein, are defined in accordance with the pertinent art and relate to enzymes capable of cutting nucleic acids by cleaving the phosphodiester bond within a polynucleotide chain. In accordance with the present invention, the nuclease is specific for the target sequence. Accordingly, the nuclease specifically binds to the target sequence and introduces a double strand break within the target sequence. Preferably, the binding site of the nuclease is up to 500 nucleotides, such as up to 250 nucleotides, up to 100 nucleotides, up to 50 nucleotides, up to 25 nucleotides, up to 10 nucleotides such as up to 5 nucleotides upstream (i.e. 5') or downstream (i.e. 3') of the nucleotide(s) that is/are modified in accordance with the present invention. The term "specifically binds", in accordance with the present invention, means that the nuclease is designed such that statistically it only binds to a particular sequence and does not bind to an unrelated sequence elsewhere in the genome. Methods for testing the DNA-binding specificity of a nuclease are known to the skilled person and include, without being limiting, phage display selection methods and the bacterial two hybrid system (Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S. 2005. Nucleic Acids Res 2005; 33:5978-5990).

Such nucleases include site-specific nucleases as well as fusion proteins comprising a DNA- binding domain and a non-specific cleavage domain of a nuclease. Site-specific nucleases include any known restriction enzymes with a known target sequence, such as e.g. BamHI, Sail etc.. In fusion proteins comprising a DNA-binding domain and a non-specific cleavage domain of a nuclease, the fusion protein specifically binds within the target sequence and brings the cleavage domain of the nuclease into the correct position for introducing a double strand break within the target sequence.

Preferably, the nuclease for use in such fusion proteins is a type II S restriction endonuclease, such as for example Fokl, Alwl, SfaNI, Sapl, Plel, NmeAIII, Mboll, Mlyl, Mmel, HpYAV, Hphl, Hgal, Faul, Earl, Ecil, BtgZI, CspCI, BspQI, BspMI, BsaXI, Bsgl, Bsel, BpuEI, Bmrl, Bcgl, Bbvl, Bael, Bbsl, Alwl, or Acul; or a type III restriction endonuclease (e.g. EcoPI I, EcoP15l, Hinflll); or is the novel restriction endonuclease termed "Clo051 " as disclosed in EP application 11 00 4635.6 and shown as SEQ ID NO: 17. More preferably, the restriction endonuclease is Fokl or Clo051.

Fokl is a bacterial type IIS restriction endonuclease. It recognises the non-palindromic penta- deoxyribonucleotide 5'-GGATG-3': 5'-CATCC-3' in duplex DNA and cleaves 9/13 nucleotides downstream of the recognition site. Fokl does not recognise any specific-sequence at the site of cleavage. The cleavage domain of Fokl is shown in SEQ ID NO: 21. Clo051 is derived from the genome of Clostridium spec. 7_2_43FAA (NCBI Reference Sequence: ZP_05132802.1 ; publication/database release date: June 9, 2010), and is particularly preferred as endonuclease in those embodiments employing the DNA-binding domain of a zinc-finger proteins or TAL effector protein, most preferably a TAL effector protein. The cleavage domain of Clo051 is shown in SEQ ID NO: 22.

Accordingly, in a preferred embodiment, the non-specific cleavage domain of a nuclease has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO:22.

Once the DNA-binding domain is anchored at the recognition site, a signal is transmitted to the endonuclease domain and cleavage occurs. The distance of the cleavage site to the DNA-binding site of the fusion protein depends on the particular endonuclease present in the fusion protein. For example, the zinc-finger nucleases employed in the examples of the present invention cleaves in the middle of a 6 bp sequence that is flanked by the two binding sites of the zinc-finger proteins and the TAL-nuclease employed in the example of the present invention cleaves in the middle of a 17 bp sequence that is flanked by the two binding sites of the TAL proteins. As a further example, naturally occurring endonucleases such as Fokl and EcoP15l cut at 9/13 and 27 bp distance from the DNA binding site, respectively. The fusion protein employed in the method of the invention retains or essentially retains the enzymatic activity of the native (restriction) nuclease. In accordance with the present invention, (restriction) nuclease function is essentially retained if at least 60% of the biological activity of the nuclease activity are retained. Preferably, at least 75% or at least 80% of the nuclease activity are retained. More preferred is that at least 90% such as at least 95%, even more preferred at least 98% such as at least 99% of the biological activity of the nuclease are retained. Most preferred is that the biological activity is fully, i.e. to 100%, retained. Also in accordance with the invention, fusion proteins having an increased biological activity compared to the endogenous nuclease, i.e. more than 100% activity. Methods of assessing biological activity of (restriction) nucleases are well known to the person skilled in the art and include, without being limiting, the incubation of a nuclease with recombinant DNA and the analysis of the reaction products by gel electrophoresis (Bloch KD.; Curr Protoc Mol Biol 2001 ; Chapter 3:Unit 3.2).

In accordance with the present invention, it is preferred that the nuclease specific for the target sequence is such a fusion protein comprising a DNA-binding domain and a nonspecific cleavage domain of a nuclease.

Preferably, the DNA-binding domain is selected from the group consisting of helix-loop-helix binding proteins, basic leucine zipper (bZip) proteins, zinc-finger proteins or TAL effector proteins. More preferably, the DNA-binding domain is selected from the group consisting of zinc-finger proteins and TAL effector proteins.

The family of helix-loop-helix binding proteins comprises standard helix-turn-helix binding proteins such as the Escherichia coli lactose repressor or the tryptophan repressor, homeodomain proteins such as the Drosophila Antennapedia protein, paired homeodomain proteins such as the vertebrate Pax transcription factors, POU domain proteins such as the vertebrate regulatory proteins PIT-1 , OCT-1 and OCT-2, winged helix-turn-helix proteins such as the GABP regulatory protein of higher eukaryotes as well as high mobility group (HMG) domain proteins (Rohs (2010)). The proteins of the bZip family consist of a basic region which interacts with the major groove of a DNA molecule through hydrogen bonding, and a hydrophobic leucine zipper region which is responsible for dimerisation. Non-limiting examples of bZip proteins are c-fos and c- jun (Rohs (2010)). Zinc-fingers are well known in the art and have been described herein above. Zinc-finger proteins may be divided into single zinc-finger proteins and triple zinc-finger proteins. The family of single zinc-finger proteins comprises for example transcription regulating proteins like GAGA, while the family of triple zinc-finger proteins comprises for example transcription regulating proteins like Krox24, Egr1 , BKLF and SP1.

Zinc-finger motifs - also referred to herein as C₂H₂ zinc-fingers - bind DNA by inserting their ot-helix into the major groove of the DNA double helix. Each zinc-finger motif primarily binds to a triplet within the DNA substrate. Binding to longer DNA sequences is achieved by linking several of these zinc-finger motifs in tandem to form zinc-finger proteins.

The term "zinc-finger nuclease", as used in accordance with the present invention, refers to a fusion protein consisting of the non-specific cleavage domain of a nuclease, preferably an endonuclease and most preferably a restriction endonuclease, and a DNA-binding domain consisting of zinc-finger motifs. The term "zinc-finger nuclease", as used herein, refers to a functional zinc-finger nuclease that essentially retains the enzymatic activity of the nuclease. The use of zinc-finger proteins for the creation of zinc-finger nucleases that recognize and cleave a specific target sequence depends on the reliable creation of zinc-finger proteins that can specifically recognize said particular target. Methods for the generation of specific zinc- finger nucleases are known to the skilled person and have been described, for example in Durai er a/. (2005)).

The term "Tal effector protein", as used herein, refers to proteins belonging to the TAL (transcription activator-like) familiy of proteins. These proteins are expressed by bacterial plant pathogens of the genus Xanthomonas. Members of the large TAL effector family are key virulence factors of Xanthomonas and reprogram host cells by mimicking eukaryotic transcription factors. The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL effector proteins from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus. PthXol , a TAL effector protein of a Xanthomonas rice pathogen, activates expression of the rice gene Os8N3, allowing Xanthomonas to colonise rice plants. TAL effector proteins are characterized by a central domain of tandem repeats, i.e. a DNA-binding domain as well as nuclear localisation signals (NLSs) and an acidic transcriptional activation domain. Members of this effector family are highly conserved and differ mainly in the amino acid sequence of their repeats and in the number of repeats. The number and order of repeats in a TAL effector protein determine its specific activity. These repeats are referred to herein as "TAL effector motifs". One exemplary member of this effector family, AvrBs3 from Xanthomonas campestris pv. vesicatoria, contains 17.5 repeats and induces expression of UPA (upregulated by AvrBs3) genes, including the Bs3 resistance gene in pepper plants (Kay, et al. (2005); Kay and Bonas (2009)). The repeats of AvrBs3 are essential for DNA binding of AvrBs3 and represent a distinct type of DNA binding domain. The mechanism of sequence specific DNA recognition has been elucidated by recent studies on the AvrBs3, Hax2, Hax3 and Hax4 proteins that revealed the TAL effectors' DNA recognition code (Boch et al. (2009)).

Tal effector motifs or repeats are 32 to 34 amino acid protein sequence motifs. The amino acid sequences of the repeats are conserved, except for two adjacent highly variable residues (at positions 12 and 13) that determine specificity towards the DNA base A, G, C or T. In other words, binding to DNA is mediated by contacting a nucleotide of the DNA double helix with the variable residues at position 12 and 13 within the Tal effector motif of a particular Tal effector protein (Boch et al. (2009).Therefore, a one-to-one correspondence between sequential amino acid repeats in the Tal effector proteins and sequential nucleotides in the target DNA was found. Each Tal effector motif primarily recognizes a single nucleotide within the DNA substrate. For example, the combination of histidine at position 12 and aspartic acid at position 13 specifically binds cytidine; the combination of asparagine at both position 12 and position 13 specifically binds guanosine; the combination of asparagine at position 12 and isoleucine at position 13 specifically binds adenosine and the combination of asparagine at position 12 and glycine at position 13 specifically binds thymidine. Binding to longer DNA sequences is achieved by linking several of these Tal effector motifs in tandem to form a "DNA-binding domain of a Tal effector protein". Thus, the term "DNA-binding domain of a Tal effector protein" relates to DNA-binding domains found in naturally occurring Tal effector proteins as well as to DNA-binding domains designed to bind to a specific target nucleotide sequence as described in the examples below.

Preferably, the DNA-binding domain is derived from the Tal effector motifs found in naturally occurring Tal effector proteins, such as for example Tal effector proteins selected from the group consisting of AvrBs3, Hax2, Hax3 or Hax4 (Bonas et al. (1989); Kay et al. (2005)).

The term "TAL-nuclease", as used in accordance with the present invention, refers to a fusion protein consisting of the non-specific cleavage domain of a nuclease, preferably an endonuclease, more preferably a restriction endonuclease, and a DNA-binding domain consisting of TAL effector motifs. The term "TAL-nuclease", as used herein, refers to a functional TAL-nuclease that essentially retains the enzymatic activity of the (restriction) endonuclease.

It is presently believed that the mechanism of double-strand cleavage by zinc-finger and TAL-nucleases requires dimerisation of the nuclease domain in order to cut the DNA substrate (Durai et al. (2005)). Dimerisation of zinc-finger nucleases or TAL-nucleases can result in the formation of homodimers if only one type of zinc-finger or TAL-nuclease is present or in the formation of heterodimers, when different types of zinc-finger or TAL- nucleases are present. It is preferred in accordance with the present invention that at least two different types of zinc-finger or TAL-nucleases having differing zinc-finger or TAL effector motifs are introduced into the oocyte. The at least two different types of zinc-finger or TAL- nucleases can be introduced into the oocyte either separately or together. Also envisaged herein is a zinc-finger or TAL-nuclease, which is provided as a functional dimer via linkage of two subunits of identical or different zinc-finger or TAL-nucleases prior to introduction into the oocyte.

Preferably, the zinc-finger or TAL-nuclease comprises at least four zinc-finger or TAL effector motifs. In the case of zinc-finger or TAL-nucleases consisting of dimers as described above this means that each monomer of the zinc-finger or TAL-nuclease comprises at least two zinc-finger or TAL effector motifs. More preferably, the zinc-finger or TAL-nuclease comprises at least six zinc-finger or TAL effector motifs, such as for example at least eight or at least ten zinc-finger or TAL effector motifs.

In accordance with the method of the invention, the nuclease that specifically binds to and introduces a double-strand break in said target sequence may also be introduced in form of a nucleic acid molecule encoding said nuclease. It will be appreciated that the nucleic acid molecule encodes said nuclease in expressible form such that expression in the oocyte results in a functional nuclease. Means and methods to ensure expression of a functional polypeptide are well known in the art. For example, the coding sequences may be comprised in a vector, such as for example a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g. in genetic engineering. The coding sequences inserted in the vector can e.g. be synthesized by standard methods, or isolated from natural sources. The coding sequences may further be ligated to transcriptional regulatory elements and/or to other amino acid encoding sequences. Such regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally regulatory elements ensuring termination of transcription and stabilisation of the transcript. Non-limiting examples for regulatory elements ensuring the initiation of transcription comprise a translation initiation codon, enhancers such as e.g. the SV40-enhancer, insulators and/or promoters, such as for example the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, chicken beta-actin promoter, CAG-promoter (a combination of chicken beta-actin promoter and cytomegalovirus immediate-early enhancer), the gai10 promoter, human elongation factor la-promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoter, the lacUV5 promoter, the autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or a globin intron in mammalian and other animal cells. Non-limiting examples for regulatory elements ensuring transcription termination include the V40-poly-A site, the tk-poly-A site or the SV40, lacZ or AcMNPV polyhedral polyadenylation signals, which are to be included downstream of the nucleic acid sequence of the invention. Additional regulatory elements may include translational enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Moreover, elements such as origin of replication, drug resistance gene or regulators (as part of an inducible promoter) may also be included. Nucleic acid molecules encoding said nuclease include DNA, such as cDNA or genomic DNA, and RNA. Preferably, embodiments reciting "RNA" are directed to mRNA. Furthermore included is genomic RNA, such as in case of RNA of RNA viruses.

It will be readily appreciated by the skilled person that more than one nucleic acid molecule may encode a nuclease in accordance with the present invention due to the degeneracy of the genetic code. Degeneracy results because a triplet code designates 20 amino acids and a stop codon. Because four bases exist which are utilized to encode genetic information, triplet codons are required to produce at least 21 different codes. The possible 4³ possibilities for bases in triplets give 64 possible codons, meaning that some degeneracy must exist. As a result, some amino acids are encoded by more than one triplet, i.e. by up to six. The degeneracy mostly arises from alterations in the third position in a triplet. This means that nucleic acid molecules having different sequences, but still encoding the same nuclease, can be employed in accordance with the present invention. The method of the present invention further comprises introducing a single stranded oligodesoxynucleotide (ODN) into the oocyte.

The term "oligodesoxynucleotide (ODN)" relates to a nucleic acid polymer made up of a sequence of desoxynucleotide residues. An ODN in accordance with the present invention is at least 30 nucleotides in length, such as e.g. at least 40 nucleotides in length, e.g. at least 50 nucleotides in length, such as e.g. at least 60 nucleotides in length, more preferably at least 70 nucleotides in length, such as e.g. at least 80 nucleotides in length, e.g. at least 90 nucleotides in length and even more preferably at least 100 nucleotides in length, such as e.g. at least 110 nucleotides in length, e.g. at least 120 nucleotides in length, e.g. at least 130 nucleotides in length, such as at least 140 nucleotides in length and most preferably at least 150 nucleotides in length. It is further preferred that the ODN in accordance with the present invention is less than 500 nucleotides in length, such as e.g. less than 400 nucleotides in length, e.g. less than 300 nucleotides in length and most preferably less than 200 nucleotides in length.

Moreover, the oligodesoxynucleotide in accordance with the present invention is a single- strand ODN (ssODN), i.e. it is not hybridised with a second, different (i.e. complementary or partially complementary) oligonucleotide strand. Nonetheless, it will be appreciated that the ssODN in accordance with the present invention may fold back onto itself, thus forming a partial or complete double-stranded molecule consisting of one oligodesoxynucleotide strand. Preferably, the ssODN in accordance with the present invention does not fold back to form a partial or complete double-stranded molecule but instead is single-stranded over its entire length.

The ODN in accordance with the present invention may be of natural as well as of (semi) synthetic origin. Thus, the ODN may, for example, be a nucleic acid molecule that has been synthesised according to conventional protocols of organic chemistry. The person skilled in the art is familiar with the preparation and the use of said probes (see, e.g., Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001 )). Also in accordance with the present invention, the ODNs may be nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA and mixed polymers. They may contain additional non-natural or derivatised nucleotide bases, as will be readily appreciated by those skilled in the art. Nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include, without being limiting, phosphorothioate nucleic acid, phosphoramidate nucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA).

In accordance with the present invention, the term "donor nucleic acid sequence" refers to a single-stranded oligodesoxynucleotide that serves as a template in the process of homologous recombination and that carries the modification that is to be introduced into the target sequence. By using this donor nucleic acid sequence as a template, the genetic information, including the modifications, is copied into the target sequence within the genome of the oocyte. In non-limiting examples, the donor nucleic acid sequence can be essentially identical to the part of the target sequence to be replaced, with the exception of one nucleotide which differs and results in the introduction of a point mutation upon homologous recombination or it can consist of an additional gene previously not present in the target sequence. In accordance with the method of the present invention, the single-stranded oligodesoxynucleotide introduced into the oocyte in step (b) comprises the donor nucleic acid sequence as defined above as well as additional regions that are homologous to the target sequence

The term "regions homologous to the target sequence" (also referred to as "homology arms" herein), in accordance with the present invention, refers to regions having sufficient sequence identity to ensure specific binding to the target sequence. Preferably, the "regions homologous to the target sequence" have a sequence identity with the corresponding part of the target sequence of at least 95%, more preferred at least 97%, more preferred at least 98%, more preferred at least 99%, even more preferred at least 99.9% and most preferred 100%. The above defined sequence identities are defined only with respect to those parts of the target sequence which serve as binding sites for the homology arms. Thus, the overall sequence identity between the entire target sequence and the homologous regions of the nucleic acid molecule of step (b) of the method of the present invention can differ from the above defined sequence identities, due to the presence of the part of the target sequence which is to be replaced by the donor nucleic acid sequence.

Methods to evaluate the identity level between two nucleic acid sequences are well known in the art. For example, the sequences can be aligned electronically using suitable computer programs known in the art. Such programs comprise BLAST (Altschul et al. (1990) J. Mol. Biol. 2 5, 403), variants thereof such as WU-BLAST (Altschul and Gish (1996) Methods Enzymol. 266, 460), FASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85, 2444) or implementations of the Smith-Waterman algorithm (SSEARCH, Smith and Waterman (1981 ) J. Mol. Biol., 147, 195). These programs, in addition to providing a pairwise sequence alignment, also report the sequence identity level (usually in percent identity) and the probability for the occurrence of the alignment by chance (P-value).

The NCBI BLAST algorithm is preferably employed in accordance with this invention. The BLASTN program for nucleic acid sequences uses as default a word length (W) of 1 1 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff, Proc. Natl. Acad. Sci., 1989, 89:10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. Accordingly, all the nucleic acid molecules having the prescribed function and further having a sequence identity of at least 95% as determined with the NCBI BLAST program fall under the scope of the invention.

Preferably, at least two regions homologous to the target sequence are present in the single- stranded oligodesoxynucleotide of (b). In accordance with the method of the present invention, the steps of introducing a nuclease into the oocyte and of introducing a single-stranded oligodesoxynucleotide into the oocyte are either carried out concomitantly, i.e. at the same time or are carried out separately, i.e. individually and at different time points. When the steps are carried out concomitantly, both the nuclease and the single-stranded oligodesoxynucleotide can be administered in parallel, for example using two separate injection needles or can be mixed together and, for example, be injected using one needle.

By introducing both a nuclease and a single-stranded oligodesoxynucleotide in accordance with the method of the invention, homologous recombination at the target sequence with the donor nucleic acid sequence is induced.

The term "homologous recombination", is used according to the definitions provided in the pertinent art. Thus, it refers to a mechanism of genetic recombination in which two DNA strands comprising similar nucleotide sequences exchange genetic material. Cells use homologous recombination during meiosis, where it serves to rearrange DNA to create an entirely unique set of haploid chromosomes, but also for the repair of damaged DNA, in particular for the repair of double strand breaks. The mechanism of homologous recombination is well known to the skilled person and has been described, for example by Paques and Haber (Paques F, Haber JE.; Microbiol Mol Biol Rev 1999; 63:349-404)

In accordance with the present invention it was surprisingly found that it is possible to introduce targeted gene modifications into the genome of vertebrate, in particular non-human mammalian and avian zygotes when employing a single stranded oligodesoxynucleotide (ODN) as donor sequence. As is shown in the appended examples, a mouse mutant harbouring a pre-planned codon replacement was successfully generated in a single step by gene targeting in one-cell embryos. As compared to the genetic manipulation of ES cells, gene targeting in one-cell embryos represents a straightforward approach that directly results in founder animals that can be used to establish a mutant colony. One-cell embryo gene targeting does not require the incorporation of selection marker genes into targeting vectors which is an essential component of targeting vectors used for ES cell engineering. In addition, mutants established from ES cells require an additional breeding step for removal of the selection marker by Flp recombinase in order to avoid its interference with the function of the targeted gene (Kwan (2002); Friedel et al. (2011 )).

By microinjecting a nuclease and a single stranded oligodesoxynucleotide as donor sequence into one-cell embryos, a G19V chocolate mutation was successfully introduced into the mouse Rab38 gene. The small GTP-binding protein RAB38 is predominantly expressed in melanocytes and retinal pigment epithelial cells and is localized to pigmented melanosomes. In chocolate mutant mice (Rab38cht), the Rab38 gene exhibits a nucleotide replacement within codon 19 (Loftus et al. (2002). The resulting G19V amino acid substitution impairs the sorting of the tyrosinase-related protein 1 (TYRP1 ) into melanosomes. In homozygous Rab38cht mice, the reduced amount of correctly located TYRP1 leads to impaired pigment production and a chocolate-like brown coat colour (Lopes et al. (2007); Wasmeier et al. (2006)). Since mutations affecting melanocyte function in humans cause oculocutaneous albinism (OCD), such as Hermansky-Pudlak syndrome, the Rab38 gene provides a candidate locus in OCD patients (DiPietro and Dell'Angelica (2005)). Upon transfer of the injected embryos, several founder mice harbouring the targeted mutation were obtained. The founders transmitted the mutant allele to their offspring, indicating that the manipulation did not interfere with fertility.

To the inventors' best knowledge, successful homologous recombination making use of a single stranded oligodesoxynucleotide as donor sequence has not been achieved so far in vertebrate oocytes, and in particular in non-human mammalian or avian oocytes. Chen et al. (201 1 ) described the use of single-stranded oligonucleotides for the modification of the eukaryotic cell lines K562, HCT1 16, U20S, A549, HEK293, HepG2 and MCF7. However, a similar approach for oocytes has not been described nor suggested in the art. In view of the numerous differences between somatic cells and oocytes, it was surprising to find that traditional approaches for the generation of precisely targeted mutations, such as the use of gene targeting vectors as templates for the homologous recombination-mediated transfer of pre-planned sequence modifications into the genome, could successfully be replaced by single-stranded ODNs. In the last few years, the knowledge regarding the requirements for successful homologous recombination when using a gene targeting vector and the repair mechanisms that mediate this effect has expanded significantly. However, the repair mechanisms underlying homologous recombination using oligonucleotides as the donor sequence is not known so far and it was surprising to find that such a mechanism is present in oocytes at all.

Typical gene targeting vectors are plasmid-based constructs that include 4 to 10 kb homology regions derived from the target locus to guide the insertion of a desired mutation during the homologous recombination process. The construction of gene targeting vectors is commonly a low throughput and time consuming task. In addition, the large size of these DNA constructs limits the number of molecules that can be introduced into embryos without eliciting toxicity. Both of these limitations are bypassed by the use of single stranded oligodesoxynucleotides as sequence specific repair templates. As is shown in the appended examples, experiments carried out with an ODN targeting the Rab38 gene indicate that these molecules successfully serve as repair template in one-cell embryos, enabling nucleotide replacements. This simplified targeting approach expedites the generation of mutant alleles as compared to the time consuming construction of plasmid vectors. Since ODNs are also able to induce in vitro sequence insertions and deletions, such modifications will further expand the utility of ODN-directed gene targeting in embryos.

In another preferred embodiment of the method of the invention, the oocyte is a fertilised oocyte.

The term "fertilised oocyte" refers to an oocyte after fusion with the fertilizing sperm. For a period of many hours (such as up to 18 hours in mice) after fertilisation, the oocyte is in a double-haploid state, comprising one maternal haploid pronucleus and one paternal haploid pronucleus. After migration of the two pronuclei together, their membranes break down, and the two genomes condense into chromosomes, thereby reconstituting a diploid organism. Preferably, the oocyte used in the method of the present invention is a fertilised oocyte in the double-haploid state.

In a further preferred embodiment of the method of the invention, the nuclease and the single stranded oligodesoxynucieotide are introduced into the oocyte by microinjection.

Microinjection into an oocyte can be carried out by injection into the nucleus (prior to fertilisation), the pronucleus (i.e. after fertilisation) and/or by injection into the cytoplasm (both before and after fertilisation). When a fertilised oocyte is employed, injection into the pronucleus is carried out either for one pronucleus or for both pronuclei. Injection of the nuclease or of a DNA encoding the nuclease of step (a) of the method of the present invention is preferably into the nucleus/pronucieus, while injection of an mRNA encoding the nuclease of step (a) is preferably into the cytoplasm. Injection of the single stranded oligodesoxynucieotide of step (b) is preferably into the nucleus/pronucieus. Preferably, the microinjection is carried out by injection into both the nucleus/pronucieus and the cytoplasm. For example, the needle can be introduced into the nucleus/pronucieus and a first amount of the nuclease and/or the nucleic acid molecule encoding said nuclease and/or the single stranded oligodesoxynucieotide is injected into the nucleus/pronucieus. While removing the needle from the oocyte, a second amount of the respective molecules is injected into the cytoplasm.

Methods for carrying out microinjection are well known in the art and are described for example in Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003. Manipulating the Mouse Embryo. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press) as well as in the examples herein below. In another preferred embodiment, the nucleic acid molecule encoding the nuclease is mRNA.

In a further preferred embodiment of the method of the invention, the regions homologous to the target sequence are localised at the 5' and 3' end of the donor single stranded oligodesoxynucleotide.

In accordance with this preferred embodiment, the donor nucleic acid sequence is flanked by the two regions homologous to the target sequence such that the single stranded oligodesoxynucleotide used in the method of the present invention consists of a first region homologous to the target sequence, followed by the donor nucleic acid sequence and then a second region homologous to the target sequence.

In a further preferred embodiment of the method of the invention, the regions homologous to the target sequence comprised in the single stranded oligodesoxynucleotide have a length of at least 15 bp each. More preferably, the regions each have a length of at least 20 bp, such as e.g. at least 30 bp, at least 50 bp at least 60 bp, at least 75 bp, more preferably at least 100 bp, such as at least 150 bp, even more preferably at least 200 bp and most preferably at least 250 bp.

In another preferred embodiment of the method of the invention, the modification of the target sequence is selected from the group consisting of substitution, insertion and deletion of a least one nucleotide of the target sequence. Preferred in accordance with the present invention are substitutions, for example substitutions of 1 to 3 nucleotides and insertions of exogenous sequences, such as loxP sites (34 nucleotides long). In a further preferred embodiment of the method of the invention, the oocyte is from a non- human mammal, an avian, a fish or a frog.

In a more preferred embodiment, the oocyte is from a non-human mammal selected from the group consisting of rodents, dogs, felids, primates, rabbits, pigs, and ruminants; or wherein the oocyte is from an avian selected from the group consisting of chickens, turkeys, pheasants, ducks, geese, quails and ratites including ostriches, emus and cassowaries; or wherein the oocytes is from a fish selected from the group consisting of trout, salmon, tuna or herring; or wherein the oocyte is from a frog selected from the genus Xenopus.

All of the mammals, avians, fish and frogs described herein are well known to the skilled person and are taxonomically defined in accordance with the pertinent art and the common general knowledge of the skilled person.

Non-limiting examples of "rodents" are mice, rats, squirrels, chipmunks, gophers, porcupines, beavers, hamsters, gerbils, guinea pigs, degus, chinchillas, prairie dogs, and groundhogs. Preferably, the rodents are mice or rats.

Non-limiting examples of "dogs" include members of the subspecies canis lupus familiaris as well as wolves, foxes, jackals, and coyotes. Preferably, the dogs are from the subspecies canis lupus familiaris and in particular are selected from beagles or dobermans. Non-limiting examples of "felides" include members of the two subfamilies: the pantherinae, including lions, tigers, jaguars and leopards and the felinae, including cougars, cheetahs, servals, lynxes, caracals, ocelots and domestic cats.

Examples of "ruminants" include, without being limiting, cattle, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn, and nilgai. Preferably, the ruminants are selected from the group consisting of cattle, goats and sheep.

The term "primates", as used herein, refers to all monkey including for example cercopithecoid (old world monkey) or platyrrhine (new world monkey) as well as lemurs, tarsiers, apes and marmosets (Callithrix jacchus). Preferably, the primates are selected from the group consisting of marmosets as well as guenons, macaques, capuchins and squirrel monkeys. The present invention also relates to a method of producing a non-human vertebrate carrying a modified target sequence in its genome, the method comprising: (a) producing an oocyte carrying a modified target sequence in its genome in accordance with the method of the invention; (b) transferring the oocyte obtained in (a) to a pseudopregnant female host; and (c) analysing the offspring delivered by the female host for the presence of the modification. In accordance with the present invention, the term "transferring the oocyte obtained in (a) to a pseudopregnant female host" includes the transfer of the fertilised oocyte but also the transfer of pre-implantation embryos of for example the 2-cell, 4-cell, 8-cell, 16-cell and _{1 Q} blastocyst (70- to 100-cell) stage. Thus, for the method of producing a non-human vertebrate, fertilisation of the oocyte is required. Said fertilisation can occur before the modification of the target sequence in step (a) in accordance with the method of the invention, i.e. a fertilised oocyte can be used for the method of producing an oocyte carrying a modified target sequence in its genome in accordance with the invention. The fertilisation can also be carried out after the modification of the target sequence in step (a), i.e. a non- fertilised oocyte can be used for the method of producing an oocyte carrying a modified target sequence in its genome in accordance with the invention, wherein the oocyte is subsequently fertilised before transfer into the pseudopregnant female host. The fertilised oocyte can be directly transferred to the pseudopregnant female host after carrying out the method of modifying the target sequence in the genome. Alternatively, the oocyte can be kept in culture to develop to the above mentioned stages of development before transfer to the female host. Methods for transferring the oocyte to a pseudopregnant female host are well known in the art and are, for example, described in Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003. Manipulating the Mouse Embryo. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press).

The step of analysing in (c) for the presence of the modification in the offspring delivered by the female host provides the necessary information whether or not the produced non-human vertebrate carries the modified target sequence in its genome. Thus, the presence of the modification is indicative of said offspring carrying a modified target sequence in its genome whereas the absence of the modification is indicative of said offspring not carrying the modified target sequence in its genome. Accordingly, offspring carrying the genetic modification can be selected.

Methods for analysing for the presence or absence of a modification are well known in the art and include, without being limiting, assays based on physical separation of nucleic acid molecules, sequencing assays as well as cleavage and digestion assays and DNA analysis by the polymerase chain reaction (PCR). Examples for assays based on physical separation of nucleic acid molecules include without limitation MALDI-TOF, denaturating gradient gel electrophoresis and other such methods known in the art, see for example Petersen et al., Hum. Mutat. 20 (2002) 253-259; Hsia et al., Theor. Appl. Genet. 111 (2005) 218-225; Tost and Gut, Clin. Biochem. 35 (2005) 335- 350; Palais et al., Anal. Biochem. 346 (2005) 167-175.

Examples for sequencing assays comprise without limitation approaches of sequence analysis by direct sequencing, fluorescent SSCP in an automated DNA sequencer and Pyrosequencing. These procedures are common in the art, see e.g. Adams et al. (Ed.), "Automated DNA Sequencing and Analysis", Academic Press, 1994; Alphey, "DNA Sequencing: From Experimental Methods to Bioinformatics", Springer Verlag Publishing, 1997; Ramon et al., J. Transl. Med. 1 (2003) 9; Meng et al„ J. Clin. Endocrinol. Metab. 90 (2005) 3419-3422.

Examples for cleavage and digestion assays include without limitation restriction digestion assays such as restriction fragments length polymorphism assays (RFLP assays), RNase protection assays, assays based on chemical cleavage methods and enzyme mismatch cleavage assays, see e.g. Youil et al., Proc. Natl. Acad. Sci. U.S.A. 92 ( 995) 87-91 ; Todd et al., J. Oral Maxil. Surg. 59 (2001 ) 660-667; Amar et al., J. Clin. Microbiol. 40 (2002) 446-452. It is further envisaged in accordance with the method of producing a non-human vertebrate carrying a modified target sequence in its genome that the step of analysis of successful genomic modification is carried out before transplantation into the female host. As a non- limiting example, the oocyte can be cultured to the 2-cell, 4-cell or 8-cell stage and one cell can be removed without destroying or altering the resulting embryo. Analysis for the genomic constitution, e.g. the presence or absence of the genomic modification, can then be carried out using for example PCR or southern blotting techniques. Such methods of analysis of successful genotyping prior to transplantation are known in the art and are described, for example in Peippo et al. (Peippo J, Viitala S, Virta J, Raty M, Tammiranta N, Lamminen T, Arc J, Myllymaki H, Vilkki J.; Mol Reprod Dev 2007; 74:1373-1378).

The non-human vertebrate produced by the method of the invention is, inter alia, useful to study the function of genes of interest and the phenotypic expression/outcome of modifications of the genome in such animals. It is furthermore envisaged, that e.g. non- human mammals of the invention can be employed as disease models and for testing therapeutic agents/compositions. Furthermore, the non-human vertebrates of the invention can also be used for livestock breeding.

In a preferred embodiment, the non-human vertebrate is a non-human mammal, an avian, a fish or a frog.

In a more preferred embodiment, the non-human mammal is selected from the group consisting of rodents, dogs, felids, primates, rabbits, pigs and ruminants; or wherein the avian is selected from the group consisting of chickens, turkeys, pheasants, ducks, geese, quails and ratites including ostriches, emus and cassowaries; or wherein the fish is selected from the group consisting of trout, salmon, tuna or herring; or wherein the frog is selected from the genus Xenopus. As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The above considerations apply mutatis mutandis to all attached claims. The figures show:

Figure 1. Schematic overview exemplifying the generation of mutant mice by microinjection of a gene specific nuclease and a single stranded oligodesoxynucleotide.

Oocytes, preferably fertilised oocytes, are collected from female mice and microinjected with mRNA coding for a gene specific nuclease pair (Nuc1 , Nuc2) and a single stranded oligodesoxynucleotide (Oligo) with homology to the target site including a sequence modification (mutation, square). The nuclease pair creates a double-strand break (DSB) at the sequence of interest. The oligodesoxynucleotide's homology regions serve as guide and repair template for double strand break-induced homologous recombination that also copies the mutant sequence (square) into the genome. Injected embryos are transferred to pseudopregnant females and the offspring is genotyped for the presence of the genetic modification. Positive animals are selected for further breeding to establish a mutant mouse strain.

Figure 2. Targeting of the Rab38 gene with a ZFN and a synthetic single stranded oligodesoxynucleotide.

The targeting oligonucleotide ODN^1013"^ was designed for the introduction of a silent nucleotide replacement into codon 18 of a Rab38⁺ or Rab38°^ht allele. The structure of the Rab38⁺ and the Rab38°^ht (cht) locus, of the targeted Rab38^IDG'WT allele, the location of the ZFN^{Ra 38} binding sites, of the Rab38 5^' probe and of PCR primers P-for and P-rev are shown. The positions of SexAI (S), BsaJI (B), and ApaLI (A) restriction sites and of SexAI restriction fragments are indicated. The Rab38°^ht allele (cht) differs from Rab38⁺ (+) by the presence of a SexAI site instead of a BsaJI site in the first exon.

Figure 3. Comparison of RAB38 codons 17/18/19, 30/31 and 44/45 of the Rab38⁺ allele (wildtype, WT), the Rab3S°^ht allele and the targeting ODN^{IDG WT}

Nucleotides differing from the wildtype sequence are shown in small letters. For ODN^IDG"WT the presence of a thymidine at the third position of codon 31 erases an ApaLI site.

Figure 4.

A: PCR amplification with primer pair P-for/P-rev of a 213 bp region covering the first exon of Rab38 from DNA from a C57BL/6 (B6) and a homozygous Rab38^IDG-°^ht (IDG-Cht/IDG-Cht) control mouse and pups C7.3 and C7.4 derived from Rab38⁺/Rab38°^ht embryos injected with ZFN^Rab38and targeting ODN^{IDG T}.

The Rab38 genotype was determined by digestion of the PCR products with BsaJI, Sail, or ApaLI. PCR products derived from a Rab38⁺ allele (B6) or a targeted Rab38^IDG'WT allele (recombined within codon 18) are digested into fragments of 153 bp and 60 bp; the product from Rab38^IDG'CM alleles (IDG-Cht/IDG-Cht) is resistant to BsaJI digestion. PCR products from Rab38⁺ and /?a£>38^{,DG CW} alleles are resistant to Sail digestion; the product from a Rab38^iDG'Cht allele (recombined within codon 44/45) would produce fragments of 157 bp and 56 bp. Digestion with ApaLI of PCR products from Rab38⁺ and Rab38^IDG'Cht alleles results into fragments of 115 bp and 98 bp; the product from a Rai 38^{/DG CW} allele (recombined within codon 30/31 ) is resistant to ApaLI digestion. The analysis of PCR products showed in both pups C7.3 and C7.4 the presence of an ApaLI resistant Rab38 allele and the absence of Sail sensitive alleles. Pup C7.3 but not C7.4 harbors a BsaJI resistant Rab38°^ht allele. M: 100 bp size ladder.

B. Southern blot analysis of SexAI digested genomic DNA using the Rab38 5' probe shows the presence of a Rab38°^ht allele (2.1 kb band) in a homozygous Rab38^IDG"Cht control mouse and pup C7.3 and its absence in pup C7.4. C: Southern blot analysis of ApaLI digested genomic DNA using the Rab38 5' probe shows a 6.6 kb fragment from the Rab38⁺ alleles of a C57BL/6 control mouse (B6) and a 10.5 kb band for the Rab38^IDG'Cht alleles from a homozygous Rajb38^DG-^cw control (IDG-Cht/IDG-Cht). Both pups C7.3 and C7.4 exhibit a 6.6 kb band from a Rab38⁺ allele and 10.5 kb band derived from an ApaLI resistant Rab38^IDG'WT in pup C7.3 and a Rab3 allele in pup C7.4.

Figure 5. Sequence analysis of mutant Rab38 alleles.

A: Comparison of sequences within the first exon of the Rab38^* and Rab38^cht alleles, the targeting ODN^{IDG WT} and of ApaLI resistant PCR products amplified with primers P_for and P_rev from genomic DNA of pup C7.3. The position of codon 18 and the ZFN^Rab3S binding regions are indicated. The six nucleotide differences between ODN^IDG"WT and the wild-type sequence are boxed; three nucleotide replacements transferred into the recombined Rab38 allele of pup C7.3 are marked by arrows.

B: Comparison of sequences within the first exon of the Rab38⁺ and Rab38°^ht alleles, the targeting ODN^{IDG WT} and of ApaLI resistant PCR products amplified with primers P_for and P_rev from genomic DNA of pup C13.5. The position of codon 18 and the ZFN^Rab38 binding regions are indicated. The six nucleotide differences between ODN^{IDG WT} and the wild-type sequence are boxed; three nucleotide replacements transferred into the recombined Rab38 allele of pup C13.5 are marked by arrows.

Figure 6. Sequence of the RabChtTAL1-Fok protein.

The region of 14 TAL repeats, recognizing the 14 nucleotide target sequence within exon 1 of the mouse Rab38 gene (TAL1 , Fig. 8), comprise a region of 476 amino acids that is linked to the nuclease domain of Fokl. The TAL repeat region is flanked with invariable sequences derived from the AvrBs3 TAL protein.

Figure 7. Sequence of the RabChtTAL2-Fok protein.

The region of 14 TAL repeats, recognizing the 14 nucleotide target sequence within exon 1 of the mouse Rab38 gene (TAL2, Fig. 8), comprise a region of 476 amino acids that is linked to the nuclease domain of Fokl. The TAL repeat region is flanked with invariable sequences derived from the AvrBs3 TAL protein.

Figure 8. Sequence comparison of Rab38 alleles

Comparison of the sequence of the mouse Rab38 gene exon 1 (Rab38⁺) to the targeting oligonucleotide ODN (IDG-Cht) and the mutant Rab38 allele of pup 58Rf8. The position of codon 19 and the binding regions for the RabChtTAL1-Fok (TAL1 ) and RabChtTAL2-Fok (TAL2) proteins are indicated. The ten nucleotide replacements between ODN^IDG"Cht and the wild-type sequence are boxed. Three nucleotide replacements were transferred into the recombined Rab38 allele of pup 58Rf8 and are marked by arrows. Two of these replacements modify codon 19 such that the Rab38 allele of pup 58Rf8 exhibits a chocolate (G19V) mutation.

Figure 9. Creation of a Rab38 chocolate mutation with TALENs and ODNs.

A: Insertion of a G19V mutation into the first exon of Rab38. The position of the targeting oligonucleotide (ODN^{IDG Cht}), the structure of the Rab38 wildtype (wt) locus, the targeted Rab38^IDG'Cht allele, the location of the TALEN binding sites, of the Rab38 5^'-hybridization probe, of PCR primers P-for and P-rev, of SexAI (S) and ApaLI (A) restriction sites and the size of genomic fragments are shown. B: Comparison of codons of RabSS"¹, Rab38° ^t and ODN^{IDG Cht}. Nucleotides and amino acids that deviate from wildtype are shown in small letters or underlined, repectively. C: Sequence comparison within the first exon of RabS^, ODN^{IDG" Ch} and of PCR products amplified with primers P-for and P-rev from tail DNA of founder Rc6 that includes the G19V replacement in codon 19, its pups Rc6-4, Rc6-3, Rc6-14, of founder Rc7, founder Rc1 and its pup Rc1-6, and of founders Rc2 - Rc5. The position of codon 19 and the TALEN^Rab38 binding sites are indicated, nucleotides deviating from wild-type are shown in small letters, deleted nucleotides are dashed, and sequence insertions are underlined. The genotype describes the mutant allele as a product of homologous recombination (HR) or NHEJ-associated deletion (Δ). D: Southern blot analysis of ApaLI digested tail DNA of founder Rc6 and of 19 Rc6 derived pups using the Rab38 5^"- hybridization probe. E: Southern blot analysis of SexAI digested tail DNA of founder Rc6 and of 19 Rc6 derived pups. The presence of a Rab38^IDG'Cht allele is indicated by a 2.1 kb SexAI fragment. F: Southern blot analysis of ApaLI digested tail DNA from founder Rc1 and its offspring Rc1-3 to Rc1-1 1. G: Southern blot analysis of ApaLI digested tail DNA from founders Rc2, Rc3, Rc4, Rc5 and Rc7. Founders Rc3, Rc4 and Rc5 exhibit smaller bands as expected from the sequence analysis of exon 1 (C), indicating additional deletion events. WT control - C57BL/6 DNA, Rab38^{Cht Cht} - DNA from a homozygous (ApaLI resistant) Rab38^IDG- mouse generated with ZFNs.

The examples illustrate the invention.

Example 1: Material and Methods

Zinc-finger nuclease and single stranded oligodesoxynucleotide design

The heterodimeric ZFN^Rab38 pair targets sequences (underlined) near to the site of the chocolate mutation (here WT sequence; bold) within the first exon of the mouse Rab38 gene (SEQ ID NO:1):

5^'- atgcagacacctcacaaggagcacctgtacaagctgctggtgatcggcgacctgggtgtgggcaagaccagcattatcaagcg ctatgtgc

accaaaacttctcctcgcactaccgggccaccattgqtqtqqacttcqcqctgaaggtqctccactqqqacccaqaaacaatqgt gcgcttgcagctctgggacattgctg -3^' (Fig. 5).

Expression constructs for the reported ZFNs recognition helices (Geurts et al. (2009) were embedded into a generic zinc-finger backbone based on the sequence of the ZIF268 protein (Klug (2010a); Klug (2010b); Moore, Choo, & Klug (2001 ); Moore, Klug, & Choo (2001 ) and generated by gene synthesis (Genscript, Piscataway, NJ, USA). The synthetic 580 bp fragment was ligated via Bglll and BamHI ends into the generic expression vectors pCAG- linker-Fok-EL (SEQ ID NO: 18) or pCAG-linker-Fok-KK (SEQ ID NO: 19), providing a CAG and T7 promoter region and the coding sequence of the Fok nuclease domain (EL or KK variants; Miller et al. (2007)), to derive the plasmids pCAG-RabZFN-L-Fok-KK (SEQ ID NO:2) and pCAG-RabZFN-R-Fok-EL (SEQ ID NO:3).

The targeting ODN ODN^{IDG WT}(SEQ ID NO:4), synthesized by Metabion (Martinsried, Germany), had a total length of 144 nucleotides including the IDG-WT mutation (Fig. 3) and the ZFN recognition site silent mutations (Fig. 5). An additional silent point mutation in codon 44 was included to differentiate between the Rab38^* allele and Rab38^IDQ-^m allele (Fig. 5).

TAL nuclease and single stranded oligodesoxynucleotide design

The TAL nucleases RabChtTAL1-Fokl and RabChtTAL2-Fokl target each a 14 nucleotide sequence within the first exon of the mouse Rab38 gene (SEQ ID NO:13; SEQ ID NO: 14) (Fig. 8). Expression constructs for the TAL nucleases were generated by ligation of 102 bp DNA elements (synthesized by Genscript) coding for TAL repeats that recognize the selected target sequences, following the recognition code of the TAL repeat amino acids 12 and 13 (HD > C; NN >G, NG > T; Nl > A). Ligated TAL elements were inserted into the generic expression vector pCAG-TAL-linker-IX-Fok (SEQ ID NO:20), providing a CAG and T7 promoter region, TAL repeat flanking sequences from the AvrBs3 TAL protein and the coding sequence of the Fokl nuclease domain, to derive the plasmids pCAG-dRabChtTAL1 (SEQ ID NO:8) and pCAG-dRabChtTAL2 (SEQ ID NO:9). The targeting ODN ODN^{IDG Cht} (SEQ ID NO:12), synthesized by Metabion (Martinsried, Germany), had a total length of 144 nucleotides including the IDG-Cht mutation (Fig. 8) and the TAL recognition site silent mutations (Fig. 8). Injection of Zygotes

For the preparation of ZFN^Rab38 mRNA, expression plasmids pCAG-RabZFN-L-Fok-KK and pCAG-RabZFN-R-Fok-EL were linearised and transcribed from the T7 promoter using the mMessage mMachine kit (Ambion) according to the manufacturer's instructions. For the preparation of RabChtTAL1-Fokl and RabChtXAL2-Fokl mRNA, expression plasmids pCAG- dRabChtTALI and pCAG-dRabChtTAL2 were linearised with Mlul and transcribed from the T7 promoter using the mMessage mMachine kit (Ambion) according to the manufacturers instructions.

The mRNA was further modified by the addition of a poly-A tail using the Poly(A) tailing kit and purified with MegaClear columns from Ambion. Finally the mRNA was precipitated and resolved in injection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.2). Each ZFN^Rab38 mRNA or TAL nuclease mRNA was diluted in injection buffer to a working concentration of 7.5 ng/μΙ and the mixture was stored together with the ODN^IDG"WT or the ODN^IDG-^Cht at -80°C. The targeting oligonucleotides ODN^IDG"WT and ODN^IDG"Cht were dissolved in water, dialysed against embryo-tested water (Sigma-Aldrich Inc, Cat. No. W1503) and diluted with injection buffer to a working concentration of 15 ng/μΙ.

For experiments with zinc finger nucleases, mouse zygotes were obtained by mating of homozygous Rab38°^ht males (Loftus et al. (2002)) to super-ovulated FVB female mice (Charles River Germany). For experiments with TAL nucleases, mouse zygotes were obtained by mating of C57BL/6 males to super-ovulated FVB female mice (Charles River Germany).

For super-ovulation three-week old FVB females were treated with 2.5 IU pregnant mares serum (PMS) 2 days before mating and with 2.5 IU Human chorionic gonadotropin (hCG) at the day of mating. Fertilised oocytes were isolated from the oviducts of plug positive females and microinjected in M2 medium (Sigma-Aldrich Inc Cat. No. M7167).

The zygotes were injected with a mixture of the targeting oligonucleotide ODN^IDG"WT or

_0DNIDG-Cht _{( 1 5 ng/(j!) and the Z}p_NRab38 _{m RNAs} (3 _{ng/(J| eacn and} 7 5 _{ng/u| eacn}) _{Qr the} J_A|_ nuclease mRNAs (7.5 ng/μΙ each) into one pronucleus and the cytoplasm following standard procedures (Nagy e? al. Manipulating the Mouse Embryo. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press). Microinjections were performed into the larger (male) pronucleus of fertilized oocytes whenever possible. For those zygotes in which the pronuclei showed no obvious size difference one of the male or female nuclei was randomly selected. Injected zygotes were transferred into pseudopregnant CD1 female mice and viable adult mice were obtained. With ODN^IDG"WT 120 mice were obtained from 445 transferred zygotes (27% recovery). All mice showed normal development and appeared healthy. With ODN^IDG"Chl 51 live offspring were obtained. All mice showed normal development and appeared healthy. Mice were handled according to institutional guidelines and housed in standard cages in a specific pathogen-free facility on a 12 h light/dark cycle with ad libitum access to food and water.

Preparation of genomic DNA

Genomic DNA was isolated from tail tips of mice derived from microinjected zygotes following the Wizard Genomic DNA Purification Kit (Promega) protocol. The obtained DNA pellet was dissolved in 100 μΙ 10 mM Tris-CI, pH 8.5, incubated over night at room temperature and stored for further analysis at 4°C. PCR, Digestion, and Sequence Analysis

To analyze the Rab38 alleles in a first round, the DNA from mice was amplified using the PCR primer pair P_for (SEQ ID NO:5) and P_rev (SEQ ID NO:6).

Amplification was performed using Taq polymerase (Qiagen) in 25 μΙ reactions with 35 cycles of 94°C - 40s, 58°C - 40s, 72°C - min. Afterwards the PCR products were directly digested with 10 units of restriction enzyme in a volume of 30 μΙ and loaded on an 1.5 % agarose gel. The undigested fragments, which represent the PCR product from recombined alleles, were extracted with the Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany) and directly sent for sequencing (GATC, Konstanz, Germany). The results were compared to the expected sequences using the Vector NTI software (Invitrogen).

Southern Blot Analysis

Genomic DNA from positive recombined mice was digested over-night with the appropriate restriction enzyme. Southern blot analysis was performed as described (Meyer er a/. (2010)) using the Rab38 5^' probe (SEQ ID NO:7), which was obtained by PCR amplification (P_Rab1 : 5^'-ctggaaactaaaattcaaggtgttatac-3^' (SEQ ID NO:15), P_Rab2: 5^'- T ATTCATTCACTT AACCATTTGTTC-3 ^' (SEQ ID NO: 16)) from C57BL/6N genomic DNA and purified with the Qiaquick PCR Purification Kit (Qiagen).

Example 2: Introduction of nucleotide replacements into the Rab38 gene using a zinc- finger nuclease and a synthetic, single-stranded oligodesoxynucleotide Introduction

We applied ZFN-assisted gene editing in one-cell mouse embryos to introduce nucleotide replacements into the mouse Rab38 gene. The small GTP-binding protein RAB38 is predominantly expressed in melanocytes and retinal pigment epithelial cells and is localized to pigmented melanosomes. In chocolate mutant mice (Rab38°^ht), the Rab38 gene exhibits a nucleotide replacement within codon 19 (Loftus et at. (2002)). The resulting G19V amino acid substitution impairs the sorting of the tyrosinase-related protein 1 (TYRP1 ) into melanosomes. In homozygous Rab38°^ht mice, the reduced amount of correctly located TYRP1 leads to impaired pigment production and a chocolate-like brown coat color (Lopes et a/. (2007); Wasmeier er a/. (2006)). Since mutations affecting melanocyte function in humans cause oculocutaneous albinism (OCD), such as Hermansky-Pudlak syndrome, the Rab38 gene provides a candidate locus in OCD patients (Di Pietro & Dell'Angelica (2005)). By employing a ZFN in one-cell embryos we introduced silent mutations into the first exon of Rab38 using a synthetic, single-stranded oligodesoxynucleotide (ODN) as repair template. These results demonstrate gene editing with synthetic ODNs in one-cell embryos. This technology represents a simplified tool for mutagenesis of the mouse germline to create e.g. disease models harboring disease-associated mutations.

Results

We explored whether nucleotide replacements can be introduced into the Rab38 gene by the co-injection of ZFN^Rab38 and a single-stranded synthetic oligodesoxynucleotide (ODN) instead of a plasmid-based (double-stranded) gene targeting vector. To stimulate HR at the Rab38 locus we used a ZFN pair (ZFN^Rab38) with a specific cleavage site located -50 bp downstream of codon 18 (Fig. 2; Fig. 5). For this purpose, we used ODN^{IDG WT} corresponding to 144 nucleotides of the sense sequence of the first exon of Rab38 (Fig. 2) (SEQ ID NO:4). _{O D N}IDG-WT _{was des}j_{gned t0} generate a Rab38^IDG'WT allele by introduction of a silent nucleotide replacement within codon 18 (Fig. 3). In addition, the ZFN^{Ra 38} recognition sequences were modified by silent replacements, that also remove an ApaLI site from codon 30/31 , to prevent the potential processing of targeted alleles by ZFN nucleases. In addition, another replacement creates a Sail site within codon 44/45 (Fig. 3).

The ODN^IDG"WT was co-injected with ZFN^{Ra 38} mRNAs into one-cell embryos derived from FVB female and Rab38°^ht (Loftus et al. (2002) male mice. From these injections we obtained 120 pups that were genotyped for the presence of the Rab38^IDG~WT allele by PCR amplification of a 213 bp region from the first exon of Rab38 (Fig. 2). A fully recombined Rab38^IDG-^WT allele could be distinguished from the Rab38⁺ and Rab38°^ht alleles by the absence of the ApaLI site and the presence of the Sail site in the PCR fragment. PCR _2g products from the Rab38⁺ and Rab38l^DG'WT allele can be cleaved with BsaJI into 153 bp and 60 bp fragments (Fig. 4A, sample B6) whereas product from the Rab38^cM allele is resistant to BsaJI.

The PCR products from pups C7.3 and C13.5 showed partial resistance to ApaLI and BsaJI digestion and full resistance to Sail digestion (Fig. 4A). In contrast, PCR products from Pup C7.4 showed partial resistance to ApaLI digestion, no resistance to BsaJI and full resistance to Sail digestion (Fig. 4A).

The sequence analysis of PCR products from pup C7.3 revealed a recombined Rab38^IDG'WT allele that includes the nucleotide replacement within codon 18 and the left ZFN binding site, but not the replacements in the right ZFN binding site and in codon 44 (Fig. 5A). The Southern blot analysis of SexAI digested DNA from pup C7.3 using the Rab38 5^* -probe showed the presence of an unmodified Rab38°^ht allele as indicated by a 2.1 kb band (Fig. 4B). The genotype of pup C7.3 was further analyzed by digestion with ApaLI and confirmed the presence of a Rab38^,DG~WT allele by an indicative 10.5 kb band and of the Rab38°^ht allele by a 6.6 kb band (Fig. 4C). Therefore, pup C7.3 exhibits a Rab38^IDG-^WT/Rab38 ^ht genotype suggesting that ODN^{IDG WT} recombined with the maternal Rab38⁺ allele, whereas the paternal Rab38°^hi allele was unaffected. For the Rab38^IDG'WT allele HR occurred in the sequence covering codon 18 to 31 but was terminated between the oligonucleotide^'s silent replacements in the left and right ZFN binding site (Fig. 5A).

The sequence analysis of ApaLI resistant PCR product from pup C13.5 revealed a recombined f?ajb38^{/DG lvr} allele that does not include the nucleotide replacements within codon 18 and 44 but the two replacements within the left ZFN binding site and one replacement in the right ZFN binding site (Fig. 5B). In contrast, the sequence and Southern blot analysis of DNA from pup C7.4 revealed the absence of a BsaJI resistant, Rab38° ^t allele and the loss of the ApaLI site through a 27 bp deletion (Δ) within the ZFN^Rab38 binding site (Fig. 4B, 4C). This Rab38⁺/Rab38ⁱⁱ genotype may arise by gene conversion from the maternal Rab38^* to the paternal Rab38°^ht allele, followed by a NHEJ repair associated deletion that occurred in a second binding cycle of ZFN^Rab38 to the recombined allele.

Taken together, the targeted alleles of mutants C7.3 and C13.5 each contained three nucleotide replacements distributed over a region of up to 46 bp. This result provides proof- of-principle for the creation of one or more single nucleotide replacements directly in one-cell embryos using ZFN technology and ODNs. Example 3: Introduction of nucleotide replacements into the mouse Rab38 gene using TAL nucleases and a synthetic, single-stranded oligodesoxynucleotide

We further explored whether nucleotide replacements can be introduced into the Rab38 gene by the co-injection of TAL nucleases and a single-stranded synthetic oligodesoxynucleotide (ODN). To stimulate HR at the Rab38 locus we used a TAL nuclease pair (RabChtTAL1-Fokl (SEQ ID NO:10), RabChtTAL2-Fokl (SEQ ID NO:11 ); Fig. 6, Fig. 7) with a specific cleavage site located -45 bp downstream of codon 19 (Fig. 8). For this purpose, we used ODN^{IDG Cht} (SEQ ID NO: 12) corresponding to a region of 144 nucleotides of the sense sequence of the first exon of Rab38. ODN^IDG~Cht was designed to generate a Rab38°^ht allele by introduction of two nucleotide replacements within codon 19 (Fig. 8) representing a G19V codon replacement, that also remove a BsaJI restriction site (Rab38^{IDG" Cht} allele). In addition, the TAL nuclease recognition sequences were modified by silent replacements to prevent the potential processing of targeted alleles by TAL nucleases (Fig. 8).

The ODN^{IDG Cht} was co-injected with RabChtTAL1-Fokl and RabChtTAL2-Fokl mRNAs into one-cell embryos derived from FVB female and C57BU6 male mice. From these injections we obtained 51 pups that were genotyped for the presence of the Rab38^!DGCht allele by PCR amplification of a 213 bp region from the first exon of Rab38 (Fig. 2). A recombined Rab38^IDGCht allele could be distinguished from the Rab38⁺ allele by the absence of the BsaJI site in the PCR fragment. PCR products from the Rab38⁺ allele can be cleaved with BsaJI into 153 bp and 60 bp fragments (Fig. 4A, sample B6) whereas product from the Rab38'^DG'Cht allele is resistant to BsaJI.

The PCR product from pup 58Rf8 showed partial resistance to BsaJI digestion. The sequence analysis of BsaJI resistant PCR product from pup 58Rf8 revealed a recombined Ra£>38^/DG~CW allele that includes both nucleotide replacements within codon 19 and one replacement between the TAL binding sites (Fig. 8), but not the replacements located within the TAL binding regions.

This result provides proof-of-principle for the creation of nucleotide replacements directly in one-cell embryos using TAL nucleases and ODNs.

Example 4: Introduction of nucleotide replacements into the mouse Rab38 gene using TAL nucleases and a synthetic, single-stranded oligodesoxynucleotide

Example 4 is a repetition of the experiment described in example 3. For the sake of completeness, the experimental procedure is described in detail in the following. To stimulate HR at the Rab38 locus a TAL nuclease pair (RabChtTAL1-Fokl (SEQ ID NO: 10), RabChtTAL2-Fokl (SEQ ID NO:11); Fig. 6, Fig. 7) was used with a specific cleavage site located ~45 bp downstream of codon 19 (Fig. 9).

For this purpose, as targeting molecule was used a synthetic, single-stranded oligodeoxynucleotide (ODN^{IDG"C t}) (SEQ ID No.: 12) of 144 nucleotides that covers 47 bp of the lagging strand sequence upstream of codon 19 and 94 bp of downstream sequence (Fig. 9A). ODN^IDG~Cht includes a G to T replacement at the second position of codon 19, creating a valine triplet and a SexAI restriction site, and a silent T to A exchange as an unique identifier of our targeted Rab38^IDG'Cht allele (Fig. 9B). To map the proficiency of HR in relation to the distance between the DSB site and ODN coded replacements, the TALEN^Rab38 spacer is located 40 bp downstream of codon 19 and 10 silent nucleotide replacements were included into ODN^{IDG Cht} (Fig. 9C). One of these replacements eliminates an ApaLI restriction site. _{O DN}iDG-c ^t _wgs mjcrojnjected together with TALEN^Rab38 mRNAs into one-cell embryos and the resulting offspring were analysed for gene editing events by PCR amplification of a 213 bp region covering the first exon of Rab38 (Fig. 9A). Founder mice were identified by the digestion of PCR products with ApaLI. Digestion resistant PCR products were recovered, cloned and analyzed by sequencing. In addition, founder mutants were analysed by Southern blot analysis of tail DNA using a hybridization probe located upstream of exon 1. Both, NHEJ and HR mediated repair events lead to the loss of the ApaLI site within exon 1 as indicated by a 10.5 kb ApaLI band in addition to the 6.6 kb wildtype fragment (Fig. 9A). HR events that include the G19V replacement can be specifically recognized by the presence of a diagnostic 2.1 kb SexAI band as compared to the 6.0 kb wildtype fragment. In addition, a second, invariable 10.5 kb fragment is detected by the hybridization probe for both, the Rab38 G19V and wildtype alleles (Fig. 9A). Among 1 17 pups derived from embryo injections of TALEN^Rab3S and ODN^{lDG Cht} we identified 7 mutant founders (Rc1 - Rc7) harboring 11 modified Rab38 alleles. Founder Rc6 contained, as shown by sequencing of cloned PCR products, a recombined Rab38^iDG'Cht allele that includes the G19V mutation and 6 additional nucleotide replacements from ODN^{IDG Cm} (Fig. 9C). A Southern blot analysis of Rc6 tail DNA showed the 2.1 kb SexAI fragment diagnostic for the Rab38^ID3'Cht allele and the recombinant 10.5 kb ApaLI band, besides a wildtype locus (Fig. 9D, 9E). Pups derived from matings of founder Rc6 were genotyped by Southern blot analysis of SexAI or ApaLI digested tail DNA and the sequencing of PCR products. Five pups showed the 2.1 kb SexAI band, demonstrating germline transmission of the a038^OG"Cfti allele (Fig. 9E). Pup Rc6-4 was further characterized by PCR and sequence analysis and confirmed the identity of its Rab38^IDG'Cht allele to the founder Rc6 (Fig. 9C). Furthermore, 7 pups derived from founder Rc6 showed a 10.5 kb ApaLI, but no 2.1 kb SexAI band (Fig. 9E) indicating the presence of additional, edited Rab38 alleles in the founder^'s germline. PCR and sequence analysis of „„

32

pup Rc6-3 revealed the presence of a targeted Rab38 allele that includes all nucleotide replacements within the TALEN^Rab38 binding region but excludes the replacements in codon 19. In contrast, pup Rc6-14 harbored a Rab38 allele exhibiting an in-frame deletion of 27 bp within the TALEN ^ab38 binding region (Fig. 9C). In conclusion, the germline of founder Rc6 constituted a mosaic of three mutant Rab38 loci, including two alleles that underwent HR with ODN^{IDG Cht} and one allele processed by NHEJ. In founder Rc7 the PCR and sequence analysis of tail DNA revealed a similar triple mutant genotype including a partially recombined Rab38^iDG'Cht allele (Rc7(a), Fig. 9C) as found in pup Rc6-3 and two NHEJ- processed loci that exhibit a 27 bp in-frame deletion in allele Rc7(c) and the combined deletion and insertion of 10 bp and 3bp, leading to a frameshift mutation within exonl (Rc7(b), Fig. 9C). Founders Rc1 - Rc5 harbored additional Rab38 alleles processed by NHEJ within the TALEN^Rab38 binding region. Founder Rc1 exhibited an in frame deletion of 60 bp that was germline transmitted and confirmed in pup Rc1-6 (Fig. 9C). Founders Rc2 and Rc3 harbored an identical in-frame deletion of six Rab38 codons, whereas founders Rc4 and Rc5 showed frame-shift mutations by the deletion/insertion of 10/3 bp (Rc4, identical to Rc7(b)) or the deletion of 1 bp (Rc5, Fig. 9C). The integrity of the modified Rab38 alleles in founders Rc1 - Rc7 and the offspring derived from Rc6 and Rc1 was analysed by Southern blotting of SexAI or ApaLI digested tail DNA using the 5^'-hybridization probe. In founders Rc6, Rc7, Rc1 , Rc2, and the Rc6 and Rc7 derived progeny the presence of the 10.5 kb ApaLI and of the 2.1 kb SexAI bands indicate the genomic integrity of the modified Rab38 alleles (Fig. 9D-G). In contrast, the NHEJ-processed Rab38 loci of founders Rc3, Rc4, and Rc5 exhibit a shortened, ~ 9 kb ApaLI fragment indicating the occurrence of additional sequence deletions. These results reveal four characteristic features of TALEN and ODN mediated mutagenesis in one-cell embryos: i) HR is capable of transferring ODN encoded nucleotide replacements into a TALEN target region, ii) nucleotide replacements occur preferentially in proximity to the DSB site but are found over a distance of up to 44 bp, Hi) a DSB site is either processed by HR into a targeted allele or by NHEJ repair into a variety of alleles containing undirected frame-shift or loss-of-codon mutations, iv) multiple alleles of both types may occur in a single founder and can be transmitted via the germline.

Taken together, these results provide proof-of-principle that TALEN and ODNs provide a versatile tool for the directed mutagenesis of the mouse germline. The occurence of 7 loci processed by NHEJ is equal to a mutagenesis rate of 6 %, whereas the presence of three homologous recombined alleles in two founders indicate an HR rate in the range of 2 %. References

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Claims

Claims

1. A method of producing an oocyte carrying a modified target sequence in its genome, the method comprising the steps:

(a) introducing into an oocyte a nuclease that specifically binds to and introduces a double-strand break in said target sequence or a nucleic acid molecule encoding said nuclease; and

(b) introducing a single stranded oligodesoxynucleotide into the oocyte, wherein the oligodesoxynucleotide comprises a donor nucleic acid sequence and regions homologous to the target sequence;

thereby inducing homologous recombination at the target sequence with the donor nucleic acid sequence.

2. The method of claim 1, wherein the oocyte is a fertilised oocyte.

3. The method of claim 1 or 2, wherein the nuclease and the single stranded oligodesoxynucleotide are introduced into the oocyte by microinjection.

4. The method of any one of claims 1 to 3, wherein the nucleic acid molecule encoding the nuclease is mRNA.

5. The method of any one of claims 1 to 4, wherein the nuclease is a fusion protein comprising a DNA-binding domain and a non-specific cleavage domain of a nuclease.

6. The method of claim 5, wherein the DNA-binding domain is selected from the group consisting of zinc-finger proteins and TAL effector proteins.

7. The method of claim 5 or 6, wherein the non-specific cleavage domain of a nuclease has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO:22.

8. The method of any one of claims 1 to 7, wherein the regions homologous to the target sequence are localised at the 5' and 3' end of the donor single stranded oligodesoxynucleotide.

9. The method of any one of claims 1 to 8, wherein the regions homologous to the target sequence comprised in the single stranded oligodesoxynucleotide have a length of at least 15 bp.

10. The method of any one of claims 1 to 9, wherein the modification of the target sequence is selected from the group consisting of substitution, insertion and deletion of a least one nucleotide of the target sequence.

The method of any one of claims 1 to 10, wherein the oocyte is from a non-human mammal, an avian, a fish or a frog.

The method of claim 11 , wherein the oocyte is from a non-human mammal selected from the group consisting of rodents, dogs, felids, primates, rabbits, pigs, and ruminants; or wherein the oocyte is from an avian selected from the group consisting of chickens, turkeys, pheasants, ducks, geese, quails and ratites including ostriches, emus and cassowaries; or wherein the oocytes is from a fish selected from the group consisting of trout, salmon, tuna or herring; or wherein the oocyte is from a frog selected from the genus Xenopus.

A method of producing a non-human vertebrate carrying a modified target sequence in its genome, the method comprising:

(a) producing an oocyte in accordance with any one of claims 1 to 12;

(b) transferring the oocyte obtained in (a) to a pseudopregnant female host; and

(c) analysing the offspring delivered by the female host for the presence of the modification.

The method of claim 13, wherein the non-human vertebrate is a non-human mammal, an avian, a fish or a frog.

The method of claim 14, wherein the non-human mammal is selected from the group consisting of rodents, dogs, felids, primates, rabbits, pigs and ruminants; or wherein the avian is selected from the group consisting of chickens, turkeys, pheasants, ducks, geese, quails and ratites including ostriches, emus and cassowaries; or wherein the fish is selected from the group consisting of trout, salmon, tuna or herring; or wherein the frog is selected from the genus Xenopus.