WO2016142719A1

WO2016142719A1 - Biallelic genetic modification

Info

Publication number: WO2016142719A1
Application number: PCT/GB2016/050686
Authority: WO
Inventors: William C SKARNES; Manousos KOUSTSOURAKIS
Original assignee: Genome Research Limited
Priority date: 2015-03-12
Filing date: 2016-03-11
Publication date: 2016-09-15
Also published as: GB201504223D0

Abstract

The present invention relates to a method for generating a biallelic genetic modification in the genome of a cell and screening for cells that comprise said biallelic genetic modification. Methods for generating cells with revertant wild type alleles are also provided.

Description

BIALLELIC GENETIC MODIFICATION

Field of invention

The present invention relates to a method for generating a biallelic genetic modification in the genome of a cell and screening for cells that comprise said biallelic genetic modification.

Background to the invention

CRISPR-Cas technology is a powerful tool for genome editing that can be applied to virtually any species, from viruses to plants to mammals [1] . The CRISPR-Cas system, exemplified by Cas9 from Streptococcus pyogenes, is an RNA-guided endonuclease that can be targeted to specific sequences by Watson-Crick base pairing between a guide RNA molecule (gRNA) and a 20 bp target sequence adjacent to an obligate NGG-trinucleotide protospacer adjacent motif (PAM) . Importantly, Cas9 can be re-programmed to induce a double-stranded break at any N₂₀NGG site in the genome simply by replacing the first 20bp of the guide RNA with the desired target sequence [2,3]. Double-strand breaks in eukaryotic cells are repaired by the cellular machinery in several ways which can be exploited to engineer a variety of mutant allele types. Non-homologous end- joining (NHEJ) is an error-prone repair process that joins the ends of the DNA strand, but often leads to small insertions/deletions (indels) at the site of the double -strand break [4,5]. Thus, NHEJ alleles are useful for introducing frame-shift mutations in protein coding genes. Alternatively, short single-stranded oligonucleotides with sequence similarity to the damaged site can be incorporated by a homology-driven repair (HDR) mechanism [6]. HDR has been employed for the generation of single base changes to model human genetic disease or to insert small epitope tags into genes [2,3,7,8]. Finally, double-stranded breaks dramatically stimulate homologous recombination (HR) with a donor plasmid that contains homology to the target site [4] . HR is required for the generation of more complex alleles where large DNA segments, such as reporter genes, are inserted into a locus [3] . Remarkably, modification of both copies of a gene (biallelic events) by NHEJ, HDR or HR has been observed [9-15] . Thus, programmable nucleases can be applied to study the function of genes in normal diploid cells, where both copies of the gene on homologous chromosomes must be modified to observe a phenotype.

A variety of methods using programmable nucleases have been published for engineering mutations in human pluripotent stem cells. The most efficient methods to date take advantage of NHEJ to create frameshift mutations in coding exons [11-17] . Typically, cells are transfected with plasmids expressing a nuclease [16]. Often these experiments include co-transfection of a fluorescent reporter to enrich for cells that take up plasmid DNAs using fluorescence- activated cell sorting (FACS) [16] .

A second strategy is nuclease-assisted gene targeting, where genetic modifications are introduced by HR following co-transfection of a circular donor vector and a nuclease [9] . By including a fluorescent reporter or a drug selection cassette in the donor vector, cells that take up the plasmid DNAs are enriched by FACS sorting or selection in drug and correctly targeted clones can be recovered at high efficiency.

A third, albeit less efficient, method is to introduce small nucleotide modifications by HDR. This method involves nucleofection of cells with short oligonucleotides and a nuclease [13]. Depending on the modification, oligonucleotides ranging in length from 80 to more than 140 bases are typically used [7, 8, 13, 15, 17-20] . The efficiency of incorporating the desired change is low (typically below 1%) , thus laborious approaches are needed to isolate the rare modified clone [8,21]. Recent studies have shown that nucleofection of pre-assembled Cas9 ribonucleoprotein (RNP) and DNA oligos may provide an efficient protocol for engineering point mutations in cells by HDR. In these studies, populations of cells incorporate a single-strand DNA oligo at the site of Cas9-induced double-strand break in a significant fraction of cells [22,23]. However, the genotypes of the individual clones were not characterised. All of these strategies have been used successfully in small scale experiments for the knockout of gene function or disease modelling in human iPS cells. However, the recovery of biallelic modified cell lines with these methods is either inefficient or genotyping of the clones is very laborious and not scalable to high throughput.

The present invention overcomes these problems by providing a highly efficient, high-throughput method for biallelic targeting of genes. This method may be applied at scale to generate large numbers of homozygous genetically modified cells for the study of gene function and disease modelling. Thus, the invention provides a method for systematically studying gene function.

In addition, for modelling human disease in a model cell system, such as human iPS cells, fluent methods are needed to generate isogenic pairs of cells with disease mutations and revertants as a control [12, 17-20]. Most often, human diseases are caused by single base pair changes that alter an amino acid in a protein, or small lesions that introduce a premature stop codon or splice site mutation. Depending on the mode of inheritance, either one allele or both alleles must be modified to model dominant or recessive diseases, respectively. For this application, the introduction of a drug selection cassette into the locus is not desirable, as it will have to be removed in a subsequent step to ensure correct expression of the modified allele. The present invention overcomes this problem and provides a highly efficient, high-throughput vector-free method for engineering biallelic point mutations and revertant alleles in cells at scale. Summary of the invention

The present inventors have shown that introducing pre-assembled RNA- guided endonuclease (e.g. Cas9 RNP consisting of recombinant Cas9 protein and in vitro synthesized guide RNA) and single-stranded DNA oligonucleotides into cells by nucleofection results in efficient biallelic genetic modification in the genome of the cell. The inventors have further shown that cells that are homozygous for the modification may be quickly and easily identified by high-throughput Sanger sequencing. Thus, the inventors have developed a highly efficient, vector-free method for engineering point mutations in cells at scale.

Thus, in a first aspect, the invention provides a method for generating a specific biallelic genetic modification in the genome of a cell and screening for cells that comprise said biallelic genetic modification, the method comprising:

(a) introducing a first RNA-guided endonuclease, a first guide RNA (gRNA) and a first single-stranded DNA oligonucleotide

(ssODN) into the cell, wherein the first gRNA is complementary to a target DNA sequence in the cell, and wherein the first ssODN is also complementary to the target DNA sequence, but includes at least one nucleotide modification relative to the target DNA sequence;

(b) culturing the cell under conditions that allow binding of the first RNA-guided endonuclease and gRNA to the target DNA sequence as a first RNA-guided ribonucleoprotein (RNP) complex, cleavage of the target DNA sequence by the first RNA-guided ribonucleoprotein complex to produce a specific double-stranded break, and homology-driven repair (HDR) between the target DNA sequence and the first ssODN;

(c) obtaining DNA from the cell;

(d) amplifying at least a portion of the DNA obtained from the cell by PCR using a first primer that binds upstream of the target DNA sequence and a second primer that binds downstream of the target DNA sequence, such that at least a portion of the target DNA sequence comprising the location of the at least one nucleotide modification is amplified;

(e) sequencing one or both strands of the amplified DNA by Sanger sequencing or single-molecule sequencing to obtain the sequence of the target DNA sequence;

(f) identifying a cell which gives a clean readable sequence trace; and (g) selecting a cell which is homozygous for the at least one nucleotide modification.

In one embodiment, the first RNA-guided endonuclease is Cas9.

The first gRNA may be a dual guide RNA or a single guide RNA.

The cell is preferably a eukaryotic cell, such as a mammalian cell or a plant cell. For example, the cell may be a mammalian embryonic stem cell, a mammalian induced pluripotent stem cell, a mammalian adult stem cell (e.g. a neural stem cell or an organoid stem cell), a mammalian cancer stem cell, a mammalian immortalised cell line (e.g. a HeLa cell), or a mammalian haploid or near-haploid cell line .

The at least one nucleotide modification in the first ssODN relative to the target DNA seguence may, for example, be (a) substitution of at least one nucleotide, (b) deletion of at least one nucleotide, or (c) insertion of at least one nucleotide.

The at least one nucleotide modification in the first ssODN relative to the target DNA sequence may be a single nucleotide substitution, such that the method generates a single point mutation in the genome of the cell. Alternatively, there may be multiple nucleotide modifications in the first ssODN relative to the target DNA sequence, such that the method generates multiple nucleotide modifications in the genome of the cell. For example, there may be multiple nucleotide substitutions in the first ssODN relative to the target DNA sequence, such that the method generates multiple point mutations in the genome of the cell. In one embodiment, there are at least two nucleotide modifications in the first ssODN relative to the target DNA sequence. When there are multiple nucleotide modifications in the first ssODN relative to the target DNA sequence, these may be adjacent to each other. The first ssODN may include at least one silent mutation to prevent re-cleavage of the target DNA sequence following incorporation of the first ssODN into the genome of the cell by homology driven repair (HDR) . In one embodiment, the at least one nucleotide modification introduces a premature stop codon. The at least one nucleotide modification may, for example, be in an exon, in a splice site, in a regulatory sequence, such as a promoter or an enhancer, or in a non-coding RNA sequence, such as a microRNA or a long non-coding RNA. The method may further comprise selecting a cell which is heterozygous for the at least one nucleotide modification.

Following selection of a cell which is homozygous or heterozygous for the nucleotide modification, the method of the invention may further comprise the following steps:

(h) introducing a second RNA-guided endonuclease, a second gRNA and a second ssODN into the cell, wherein the second gRNA is complementary to the modified target DNA sequence and wherein the second ssODN is complementary to the modified target DNA sequence but includes at least one nucleotide modification relative to the modified target DNA sequence;

(i) culturing the cell under conditions that allow binding of the second RNA-guided endonuclease and gRNA to the modified target DNA sequence as a second RNA-guided ribonucleoprotein (RNP) complex, cleavage of the modified target DNA sequence by the second RNA- guided RNP to produce a specific double-stranded break, and homology-driven repair (HDR) between the modified target DNA sequence and the second ssODN;

(j) obtaining DNA from the cell;

(k) amplifying at least a portion of the DNA obtained from the cell by PCR using a first primer that binds upstream of the target DNA sequence and a second primer that binds downstream of the target DNA sequence, such that at least a portion of the target DNA sequence containing the at least one nucleotide modification is amplified; (1) sequencing one or both strands of the amplified DNA by Sanger sequencing (e.g. dye-terminator Sanger sequencing) or single- molecule sequencing to obtain the sequence of the target DNA sequence;

(m) identifying a cell which gives a clean readable sequence trace; and

(n) selecting a cell which is homozygous or heterozygous for the at least one nucleotide modification relative to the modified target DNA sequence .

In one embodiment, the second ssODN may be complementary to the wild-type target DNA sequence and step (n) may comprise the step of selecting a cell which comprises the wild type target sequence, thereby identifying a cell that has a biallelic revertant phenotype.

In this embodiment, the cell selected in step (n) may be tested for function of the wild type target DNA sequence to confirm that function has been restored in the cell.

This allows the generation and testing of revertants to provide confirmation that any phenotypic changes observed with cells homozygous for the base pair modification are due to the modification itself, rather than any other additional change (s) in the genome .

The second ssODN may also introduce a silent mutation to differentiate between true reversion events or possible wild-type contamination .

In this aspect of the invention, the at least one nucleotide modification relative to the target DNA sequence present in the first ssODN used in step (a) must not perturb recognition of the PAM site .

In an alternative embodiment, the method of the invention may further comprise the following steps:

(h) introducing a second RNA-guided endonuclease and a second gRNA into a cell which is heterozygous for the at least one nucleotide modification relative to the modified target DNA sequence, wherein the second gRNA is complementary to the modified target DNA sequence;

(i) culturing the cell under conditions that allow binding of the second RNA-guided endonuclease and gRNA to the modified target DNA sequence as a second RNA-guided ribonucleoprotein (RNP) complex, cleavage of the modified target DNA sequence by the second RNA- guided RNP to produce a specific double-stranded break, and gene conversion with the corresponding heterozygous wild type allele;

(j) obtaining DNA from the cell;

(k) amplifying at least a portion of the DNA obtained from the cell by PCR using a first primer that binds upstream of the target DNA sequence and a second primer that binds downstream of the target DNA sequence, such that at least a portion of the target DNA sequence containing the at least one nucleotide modification is amplified;

(1) sequencing one or both strands of the amplified DNA by Sanger sequencing (e.g. dye-terminator Sanger sequencing) or single- molecule sequencing to obtain the sequence of the target DNA sequence;

(m) identifying a cell which gives a clean readable sequence trace; and

(n) selecting a cell which is homozygous for the wild type target DNA sequence. The cell selected in step (n) may be tested for function of the wild type target DNA sequence to confirm that function has been restored in the cell.

This embodiment allows the generation and testing of revertants to provide confirmation that any phenotypic changes observed with cells heterozygous for the base pair modification are due to the modification itself, rather than any other additional change (s) in the genome . In one embodiment of either of the alternative embodiments described above, the second RNA-guided endonuclease is Cas9. The second gRNA may be a dual guide RNA or a single guide RNA.

In one embodiment, the method comprises introducing at least two first RNA-guided endonucleases, at least two gRNAs and at least two ssODNs into the cell, such that at least two target DNA sequences are modified by the method.

Each of the embodiments described above may be combined with any one or more of any other embodiments.

In another aspect, the invention provides a cell produced by the method of the invention. In a further aspect, the invention provides a kit of parts for carrying out the method of the invention. For example, the invention provides a kit comprising (i) an RNA-guided endonuclease, (ii) a gRNA, (iii) a first ssODN, (iv) a first PCR primer, (v) a second PCR primer, and (vi) a DNA polymerase, wherein the first gRNA is complementary to a target DNA sequence to be modified in the genome of a cell, the first ssODN is also complementary to the target DNA sequence, but includes at least one nucleotide modification relative to the target DNA sequence, the first PCR primer binds upstream of the target DNA sequence and the second primer binds downstream of the target DNA sequence.

These and other aspects of the invention are described in further detail below. Brief description of drawings

Figure 1 shows the oligonucleotide design of ssODN for the introduction of an Xbal site into exon 5 of human TP53 with Cas9 RNP. Base pair substitutions relative to the human reference sequence (GRCh38) are shown in bold text. The protospacer adjacent domain (PAM) of the CRISPR site is underlined. Figure 2 shows the possible genotypes of single clones. For mutations that introduce base pair substitutions, Sanger sequencing will produce clean, readable traces for the genotypes of interest (wt/wt; mutant/wild-type; mutant/mutant) . Insertions or deletions of the allele by non-homologous end joining (NHEJ) repair will vary in size and therefore produce unreadable sequence traces. Importantly, the purity of the clone is also evident from the sequence trace. Mixed clones will produce poor quality sequence reads .

Figure 3 shows a summary of genotypes of individual targeted clones following PCR sequencing. Homology directed-repair (HDR) events produce readable sequence traces, whereas non-homologous end-joining (NHEJ) events produced mixed traces.

Figure 4 shows sequence traces across exon 5 of TP53 for (A) a homozygous and (B) a heterozygous clone. (A) Sequence traces showing biallelic modification of TP53. (B) Sequence traces showing monoallelic modification of TP53. The double peaks indicate that the cells are heterozygous for both the Xbal site and PAM site mutation. (C) Sequence traces showing biallelic incorporation of the PAM site mutation, but not the Xbal site. The arrows indicate the PAM site mutation. Figure 5 shows the effect of varying the length of single-stranded oligonucleotides for HDR. RPLP assay to detect the incorporation of the Xbal site at the TP53 locus in a population of transfected cells. A lower band (arrow) indicates cleavage of the PCR product with Xbal. Cleavage is observed in experiments were the ssODN is 80 bases or greater. Notably, cleavage is observed at similar levels using a ssODN with (lane 140) or without a PAM site mutation (lane 140+) .

Figure 6 shows fluent engineering of mutant and revertant alleles. Strategy for the generation of mutant and revertant alleles by homology-directed repair. The initial mutation is generated with Cas9 RNP containing a guide RNA (gRNA) complementary to the wild- type locus and an ssODN that contains desired mutation. The base substitutions incorporated into the target locus of the mutant cell create a new CRISPR site that is used for the reversion of mutation. The revertant is generated with Cas9 RNP containing the new guide complementary to the mutant locus and a ssODN that contains the wild-type sequence.

Figure 7 shows the design of oligonucleotides for mutant and revertant alleles. In this example, the oligonucleotide introduces a premature stop codon in exon 5 of TP53.

Figure 8 shows the design of oligonucleotides for mutant and revertant alleles. In this example, the first oligonucleotide introduces a mis-sense mutation (N308D) and two silent changes into exon 8 of the PTPN11 gene. The second oligonucleotide reverts the mutation to wild-type and introduces a novel silent mutation.

Figure 9 shows sequence verification of correctly modified (a) N308D mutant (a) and (b) wild-type revertant human iPS cell clones. The cells are heterozygous for the mutant and revertant alleles at the bases underlined below the sequence traces.

Figure 10 shows the strategy for altering the zygosity of the mutation in heterozygous mutant cells by gene conversion. In this example, cells heterozygous for the N308D mutation in exon 8 of PTPN11 are reverted to wild-type by gene conversion after inducing a double strand break in the wild-type allele with Cas9 RNP.

Figure 11 shows sequence verification of starting cells that are heterozygous for the N308D mutation and cells that homozygous for the N308D mutation following gene conversion.

Detailed description

The present invention provides a method for generating a specific biallelic genetic modification in the genome of the cell. This method includes screening for cells that comprise said biallelic genetic modification. The method includes the step of introducing a first RNA-guided endonuclease, a first guide RNA (gRNA) and a first single-stranded DNA oligonucleotide (ssODN) into the cell, wherein the first gRNA is complementary to a target DNA sequence in the cell, and wherein the first ssODN is also complementary to the target DNA sequence, but includes at least one nucleotide modification relative to the target DNA sequence.

The DNA target sequence may be any short sequence in the genome that is adjacent to an appropriate protospacer-adj acent motif (ΡΆΜ) required for target recognition and cleavage by the endonuclease [24, 25] . The PAM site for Streptococcus pyogenes Cas9, for example, is 5'-NGG or 5' NAG [25].

The RNA-guided endonuclease, gRNA and ssODN may be introduced into the cell using any method that delivers sufficient amounts of protein, RNA or DNA to a high proportion of cells. Preferably, the RNA-guided endonuclease, gRNA and ssODN are introduced in the cell by nucleofection, which results in efficient transfection of pluripotent stem cells with protein, RNA and DNA, but other methods such as lipofection and electroporation may be used. When the RNA- guided endonuclease is Cas9, a cell-permeable version of Cas9 may be used .

Any RNA-guided endonuclease may be used in the method, provided that it is active in eukaryotic cells and can be delivered to cells in the form of a recombinant protein. Preferably, the RNA-guided endonuclease is Cas9.

The gRNA may be a dual guide RNA or a single guide RNA. For example, a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) may be used as a dual guide RNA. Dual guide RNAs may be chemically synthesised, eliminating the requirement to clone, in vitro transcribe, and purify the single guide RNA. The method may be applied to any cultured eukaryotic cell (e.g. a plant cell or a mammalian cell) , provided that the cell can be transfected and single clones of cells recovered or isolated. Preferred cell types for use in the method include mammalian embryonic stem cells, mammalian induced pluripotent stem cells, mammalian adult stem cells (e.g. neural stem cells and organoid stem cells), mammalian cancer stem cells, mammalian immortalised cell lines (e.g. HeLa cells) and mammalian haploid or near-haploid cell lines .

The ssODN may be any length, within practical limits. However, the quality of the ssODN is inversely correlated with its length. Therefore, the ssODN is preferably less than 200, less than 190, less than 180, less than 170, less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, or less than 100 nucleotides in length. Most preferably, the sODN is less than 140 nucleotides in length. For the introduction of small changes (less than 5 nucleotide modifications) , 80-100 nucleotides appears to be the optimal length of the ssODN for the introduction of small changes. In cases where larger sequences are to be inserted into the target DNA sequence (e.g., an epitope tag), it is desirable to provide least 80 - 100 nucleotides of sequence complementary to the target DNA sequence plus the inserted sequence. The method allows introduction of various types of genetic modification, including a single nucleotide change (i.e. a single point mutation) that alters an amino acid in a protein, a small lesion that introduces a premature stop codon or splice site mutation. The method overcomes problems associated with the introduction of a drug selection cassette or other selectable marker, which would have to be removed in a subsequent step to ensure correct expression of the modified allele.

The at least one nucleotide modification in the first ssODN relative to the target DNA sequence may be any type of nucleotide modification. For example, the nucleotide modification may be (a) substitution of at least one nucleotide, (b) deletion of at least one nucleotide, or (c) insertion of at least one nucleotide. In this way, the method is able to generate specific modifications in the genome of the cell. For example, the at least one nucleotide modification may model a specific mutation known to occur in a particular disease. In one embodiment, the at least one nucleotide modification is able to knock-out a specific gene, e.g. by introducing a premature stop codon or a frame-shift mutation.

There may be a single nucleotide modification or multiple nucleotide modifications in the first ssODN relative to the target DNA sequence. When the at least one nucleotide modification in the first ssODN relative to the target DNA sequence is a single nucleotide substitution, the method generates a single point mutation in the genome of the cell. When there are multiple nucleotide substitutions in the first ssODN relative to the target DNA sequence, the method generates multiple point mutations in the genome of the cell.

When there are multiple nucleotide modifications in the first ssODN relative to the target DNA sequence, there may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9 or at least 10 nucleotide modifications in the ssODN relative to the target DNA sequence.

The first ssODN may include at least one silent mutation to prevent re-cleavage of the target DNA sequence following incorporation of the first ssODN into the genome of the cell by homology driven repair (HDR) . Preferably, the first ssODN includes at least two or at least three such silent mutations. A "silent" mutation is one which does not significantly alter the phenotype of the cell.

Preferably, the at least one nucleotide modification creates a new CRISPR site with minimal off-target sites in the genome. For this reason, altering the PAM-proximal sequence of the CRISPR site by at least two base pairs is optimal to prevent re-cleavage and damage to the modified site. To avoid cleavage of the modified site by persistent Cas9 activity in the cell, the ssODN should preferably contain at least two base pair changes in the seed region (12 bp upstream of the PAM site) of the CRISPR site. A single base pair change in the seed region may not be sufficient to prevent re- cleavage of the site.

When there are multiple nucleotide modifications in the first ssODN relative to the target DNA sequence, the multiple nucleotide modifications may be adjacent to each other or spaced apart. The at least one nucleotide modification may be in any coding or non-coding region of the target DNA. For example, the nucleotide modification may be in an exon, in a splice site or in a regulatory sequence, such as a promoter or enhancer. In one embodiment, the at least one nucleotide modification introduces a nonsense codon or an amino acid change in a protein-coding exon. In another embodiment, the at least one nucleotide modification is in a non-coding RNA sequence, such as a micro RNA or a long non-coding RNA. The at least one nucleotide modification is not necessarily in a functional element of the genome, but may also be in regions of the genome devoid of functional elements, e.g. for the introduction of site- specific recombination sites for chromosome engineering.

The method includes the step of culturing the cell under conditions that allow binding of the first RNA-guided endonuclease and gRNA to the target DNA sequence as a first RNA-guided ribonucleoprotein (RNP) complex, cleavage of the target DNA sequence by the first RNA- guided ribonucleoprotein complex to produce a specific double- stranded break, and homology-driven repair (HDR) between the target DNA sequence and the first ssODN. The RNA-guided endonuclease, gRNA and ssODN may be delivered to a population of cells by, for example, nucleofection . The cells are plated at clonal density and after a suitable time in culture, a single clone is picked and expanded in culture . The method includes the step of obtaining DNA from the cell. DNA may, for example, be obtained from the cell by lysing a portion of the cells of a single clone to prepare genomic DNA for subsequent PCR and sequencing. For example, the cells may be lysed in buffer containing mild-detergent and digested with Proteinase K. The remaining cells may be either kept in culture or frozen. The method includes the step of amplifying at least a portion of the DNA obtained from the cell by PCR using a first primer that binds upstream of the target DNA sequence and a second primer that binds downstream of the target DNA sequence, such that at least a portion of the target DNA sequence comprising the location of the at least one nucleotide modification is amplified. The location of the PCR primers upstream and downstream of the target DNA sequence means that this target sequence is amplified. In this way, any nucleotide modifications in the target sequence are also amplified. The PCR product should preferably contain a sufficient amount of flanking DNA sequence to permit sequencing of the at least one nucleotide modification .

The method includes the step of sequencing one or both strands of the amplified DNA by Sanger sequencing, for example dye-terminator Sanger sequencing, or single-molecule sequencing [21] to obtain the sequence of the target DNA sequence.

The method includes the step of identifying a cell which gives a clean readable sequence trace. A clean readable sequence is one that unambiguously incorporates a single base at each nucleotide position of the sequence in homozygous wild-type and homozygous mutant cells, or incorporates one of two possible bases at the position of the modified site and a single unambiguous base at all other positions of the sequence in heterozygous mutant cells.

Only those clones that are wild-type, heterozygous or homozygous for the intended mutation yield readable sequence traces, and allow pure and mixed cell populations to be easily distinguished. Cells that are wild-type or homozygous for the nucleotide modification can then be readily distinguished as they give a clean sequence trace (see Figure 4) . From these sequence traces, cells that are homozygous for the base pair modification can then be identified. The method includes the step of selecting a cell which is homozygous for the at least one nucleotide modification. Single clones of cells may, for example, be isolated, e.g. by dilution or cell sorting and/or actively cloned. Cells with the desired modification, may be either kept in culture or frozen, and are chosen for further expansion and experimentation.

The method may further include the step of selecting a cell which is heterozygous for the at least one nucleotide modification. These cells represent good controls in functional assays that compare wild type cells with bialleic knockout cells.

Following selection of a cell which is homozygous or heterozygous for the at least one nucleotide modification, the method may further comprise introducing a further mutation into the target DNA sequence. For example, the further mutation may restore the modified target DNA sequence back to the wild-type target sequence, thus generating a revertant cell with a wild-type phenotype. This provides confirmation that any phenotypic changes observed in the mutant cell are due to the nucleotide modification. Thus, following selection of a cell which is homozygous for the at least one nucleotide modification, the method may further comprise the following steps:

(i) culturing the cell under conditions that allow binding of the second RNA-guided endonuclease and gRNA to the modified target DNA sequence as a second RNA-guided ribonucleoprotein (RNP) complex, cleavage of the modified target DNA sequence by the second RNA- guided RNP to produce a specific double-stranded break, and homology-driven repair (HDR) between the modified target DNA sequence and the second ssODN; (j) obtaining DNA from the cell;

(1) sequencing one or both strands of the amplified DNA by Sanger sequencing or single-molecule sequencing to obtain the sequence of the target DNA sequence;

(m) identifying a cell which gives a clean readable sequence trace; and

(n) selecting a cell which is homozygous or heterozygous for the at least one nucleotide modification relative to the modified target DNA sequence.

The description of each of steps (a) - (g) provided above applies equally to steps (h) - (n) and any of the particular embodiments described for steps (a) - (g) may be used in steps (h) - (n) . For example, the second RNA-guided endonuclease may be Cas9.

In this aspect of the invention, the at least one nucleotide modification relative to the target DNA sequence present in the first ssODN used in step (a) must not perturb recognition of the PAM site. This is so that Cas9 can still be targeted to the modified target DNA sequence by Watson-Crick base pairing between the gRNA and the 20 bp target sequence adjacent to the PAM site.

The cell selected in step (n) may be tested for function of the wild type target DNA sequence to confirm that function has been restored in the cell. The second ssODN may also introduce a silent mutation to differentiate between true reversion events or possible wild-type contamination. A silent mutation is one which does not significantly alter the phenotype of the cell.

(h) introducing a second RNA-guided endonuclease, such as Cas9, and a second gRNA into a cell which is heterozygous for the at least one nucleotide modification relative to the modified target DNA sequence, wherein the second gRNA is complementary to the modified target DNA sequence;

(j) obtaining DNA from the cell;

(m) identifying a cell which gives a clean readable sequence trace; and

(n) selecting a cell which is homozygous for the wild type target DNA sequence .

The cell selected in step (n) may be tested for function of the wild type target DNA sequence to confirm that function has been restored in the cell. This embodiment allows the generation of revertants by gene conversion to correct the at least one nucleotide modification and generate a cell that is homozygous for the wild type sequence. The revertant cells are then tested to provide confirmation that any phenotypic changes observed with cells heterozygous for the base pair modification are due to the modification itself, rather than any other additional change (s) in the genome. Gene conversion is the process by which one DNA sequence replaces a homologous sequence, such that the sequences become identical after the conversion event. Gene conversion may be allelic, meaning that one allele of the same gene replaces another allele or ectopic, meaning that one paralogous DNA sequence converts another. During gene conversion, genetic information is exchanged between homologous sequences, either from the same chromosome (intrachromosomal) or between homologous chromosomes (interchromosomal) . In the present invention, gene conversion is allelic and interchromosomal.

Thus, the invention provides a fluent method for modelling diseases (e.g. human diseases) in a model cell system, such as human induced pluripotent stem cells (iPS cells), and allows the generation of isogenic pairs of cells with disease mutations and revertants as a control. In addition, the invention also provides a method for systematically studying gene function. The generation and testing of revertants provides confirmation that any phenotypic changes observed with cells homozygous for the nucleotide modification are due to the modification itself, rather than any other additional change (s) in the genome. The method provided herein also allows modification of more than one site in the genome, i.e. the introduction of more than one gRNA and ssODN into the cell to alter multiple sites in the genome simultaneously (e.g. to mutate two or more genes simultaneously). This is called "multiplexing". Therefore, the method may comprise introducing at least two first gRNAs and at least two first ssODNs into the cell in step (a) , such that at least two target DNA sequences are modified by the method. In this embodiment, there are at least two pairs of a first gRNA and an ssODN, such that each pair of gRNAs and ssODNs is complementary to a different target DNA sequence . Also provided herein is a cell produced by the method of the invention .

In a further aspect, the invention provides a kit of parts for carrying out the method of the invention. For example, the kit may comprise (i) an RNA-guided endonuclease , such as Cas9, (ii) a gRNA, (iii) a first ssODN, (iv) a first PCR primer, and (v) a second PCR primer, wherein the first gRNA is complementary to a target DNA sequence to be modified in the genome of a cell, the first ssODN is also complementary to the target DNA sequence, but includes at least one nucleotide modification relative to the target DNA sequence, the first PCR primer binds upstream of the target DNA sequence and the second primer binds downstream of the target DNA sequence. The kit may further optionally comprise a DNA polymerase. EXAMPLES

1) Biallelic modification of the human TP53 gene

Human iPS cells were transfected by nucleofection with Cas9-RNP directed to exon 5 of the human TP53 gene and an oligonucleotide that introduces a three base pair change to create a novel Xbal site (see Figure 1) .

After 48 hours, single cells were seeded and 96 individual colonies were expanded for genotyping. PCR amplicons from the targeted region were sequenced by Sanger sequencing. In theory, we expect to find six genotype classes (see Figure 2), and only the genotypes of interest (homozygous wild type, homozygous mutant and heterozygous wild type/mutant) will produce clear sequence traces.

Nearly all of the clones are modified by HDR or NHEJ (only four clones gave wild-type sequence) , indicating that the delivery of Cas9 RNP into cells is nearly 100% efficient (see Figure 3) . About half of the clones (42/96) gave readable sequence traces that showed modification of the locus by HDR. Remarkably, the majority of these clones (23/42) are homozygous for the mutation, i.e., the DNA oligo modified both alleles (see Figure 3) . Fifteen of the homozygous clones have incorporated the oligonucleotide sequence with nucleotide precision and a further 8 clones are homozygous for the novel Xbal site, but have not incorporated the PAM site mutation (see Figure 3 and 4C) .

Thus, without any drug selection or enrichment steps, more than 20% of transfected clones are homozygous for the desired mutation. This experiment has been repeated with oligonucleotides ranging in size from 60 to 140 bases and 80-100 bases was determined to be optimal (see Figure 6) . In this experiment, it was shown that the PAM site mutation is not required for efficient HDR (140+ lane of the gel shown in Figure 6) . Presumably, the introduction of 3 mismatches into the modified site is sufficient to protect the locus from damage by Cas9 RNP. This method is readily scalable to high- throughput, requiring only the synthesis of an oligonucleotide and gRNA for the nucleofection of cells to generate a range of point mutations (mis-sense, non-sense, frameshift and splice site mutations) in one or both copies of the gene.

Revertant alleles are important controls for experiments using cells where there is a risk that undesired genetic changes (e.g. spontaneous mutations/rearrangements) have occurred during the modification of the cells or during culture. Here, it is shown that revertant alleles can be very simply engineered by designing a guide RNA to the modified sequence and using a wild-type DNA oligonucleotide to correct the mutation (see Figure 6) . In this application, the oligonucleotide used for modification of the locus must not contain mutations that perturb recognition of the PAM site by Cas9, otherwise the new site will not be recognized and cleaved by Cas9 RNP. 2) Generation of mutant and revertant alleles of PTPN11

As a proof-of-principle experiment, mutant and revertant alleles were generated in PTPN11, commonly mutated in Noonan Syndrome. Figure 8 shows the design of oligonucleotides for mutant and revertant alleles. In this example, the first oligonucleotide introduces a mis-sense mutation (N308D) in exon 8 of PTPN11 and two silent mutations (base pair substitutions are shown in bold) . The second oligonucleotide reverts the mutation to wild-type, introducing a novel silent mutation to differentiate between true reversion events or possible wild-type cell contamination.

Human iPS cells were transfected by nucleofection with Cas9-RNP directed to wild-type sequence of PTPNll exon 8 and an oligonucleotide (100-mer) that introduces the N308D mutation and two nearby silent mutations. The silent mutations are introduced to prevent re-cleavage of the gene after incorporation of the oligonucleotide into the genome by HDR. After the cells have been allowed to recover, single cells were seeded and 96 individual colonies were picked and expanded for genotyping. PCR amplicons from the targeted region were sequenced by Sanger sequencing.

In this experiment, 17 of 96 clones were modified by HDR or NHEJ, of which 4 clones were heterozygous for the N308D mutation and associated silent mutations (Figure 9a) . One of the N308D clones was expanded and used for a second nucleofection where Cas9 RNP directed to the mutant sequence (Figure 8) and an oligonucleotide (100-mer) containing the wild-type sequence and one novel silent mutation. Following nucleofection, single cells were plated and 96 individual colonies were picked and expanded for PCR genotyping. In this experiment, 32 of 96 clones were modified by HDR and NHEJ, of which 7 clones were heterozygous for the reversion allele (Figure 9b) .

Thus, we have demonstrated that mutant and revertant alleles can be generated fluently in two sequential targeting experiments, provided the oligonucleotide sequence used to introduce the desired mutation does not perturb recognition of the PAM-site (e.g., nucleotide change the *GG' of the ^GG' PAM site for SpCas9) . Although our experiments used oligonucleotides with at least 3 mismatches to the CRISPR site to prevent re-cleavage of the target region after incorporation of the oligonucleotide sequence into the genome, we predict that this method can work with two or one nucleotide changes, albeit at a lower efficiency. 3. Altering zygosity by gene conversion

In the reversion experiment described above for PTPN11, we noted several clones with fully wild-type sequence, suggesting that the mutation was corrected by gene conversion. Gene conversion is process where genetic information is exchanged between homologous sequences, either from the same chromosome ( intrachromosomal ) or between homologous chromosomes (interchromosomal) . We reasoned that we can take advantage of gene conversion to alter the zygosity of a mutation with Cas9 RNP. For example, nucleofection of cells with Cas9 RNP directed to the wild-type sequence can be used to convert cells that are heterozygous for a mutant allele to cells that are homozygous for the mutant allele. Conversely, nucleofection of cells with Cas9 RNP directed to the mutant allele can be used to convert cells that are heterozygous for a mutant allele to cells that are homozygous wild-type, thus reverting the mutation. In such gene conversion experiments, since the exchange of genetic information is between homologous chromosomes, there is no requirement for a single-stranded oligonucleotide template.

As a proof-of-principle experiment, we converted human iPS cells that are heterozygous for the N308D allele (described in Example 2) to cells that are homozygous for the N308D mutation. Heterozygous N308D cells were transfected by nucleofection with Cas9 RNP directed to the wild-type PTPN11 sequence (Figure 10). After the cells have been allowed to recover, single cells were seeded and 96 individual colonies were picked and expanded for genotyping. PCR amplicons from the targeted region were sequenced by Sanger sequencing.

In this experiment, we recovered one clone that was homozygous for the ND308 mutation (Figure 11) and thus had undergone gene conversion. Another 30 clones were modified by NHEJ. The ability to specifically cleave either the mutant or wild-type allele with Cas9 provides a method to alter the zygosity of either allele in a very simple manner.

METHODS

Generation of sgRNA and ssODN

Single guide RNAs (sgRNA) were in vitro transcribed following cloning of forward and reverse strand oligos into the Bsal site of pl260_T7_gRNA_BsaI (provided by Sebastian Gerety, Wellcome Trust Sanger Institute, unpublished) . Plasmids were linearized at a unique Dral site downstream of the sgRNA sequence and 4ug of ethanol precipitated vector DNA was used as template for in vitro transcription using MEGAshortscript™ T7 Transcription Kit (Life Technologies). Transcription was carried out for 4 hours at 37°C followed by column purification using MEGAclear kit (Ambion) . Eluted RNA was ethanol precipitated, washed twice with 70% ethanol and air dried at RT for 15-20 minutes in sterile conditions. RNA was resuspended in 15ul sterile PBS for 10-15 minutes at RT. A 1 in 4 dilution of the sample was used to determine the concentration of RNA and to check its quality on an agarose gel. ssODN were ordered from Integrated DNA Technologies (IDT) as PAGE purified Ultramer DNA oligos. Lyophilized oligos were resuspended in sterile conditions in an appropriate volume of PBS to have 500pmol in volume no larger than 6ul .

Culture of human iPS cells

Early passage (pl2-16) human iPS cells were routinely cultured using TeSR-E8 media (StemCell Technologies) on tissue culture plates coated with Synthemax II-SC substrate (Corning) . For passaging cells ReLeSR (StemCell Technologies) was used to disaggregate the colonies for 4-6 minutes at RT prior to scraping them off with a cell lifter and gentle trituration once or twice with a 10ml pipette.

Nucleofection of Cas9 RNP and ssODN

Transfection of cells with Cas9 RNP and oligos was carried out with a Nucleofector 4D (Lonza) . A single cell suspension was prepared by incubating cells in Accutase (Millipore) for up to 10 minutes at 37°C. An aliquot of the cell suspension was counted and 8x105 cells were resuspended in a volume of 100 ul Amaxa P3 Primary Cell buffer (4D-Nucleofecter X Kit L; Lonza) . Cas9 RNP was pre-assembled in vitro in a sterile tube by adding 2 ul recombinant Cas9 protein (10 ug/ul; ToolGen) to 2ul (7-10 ug/ul) of in vitro transcribed sgRNA and incubated at RT for at least 10 minutes. Just prior to nucleofection, 6ul (500pmol in PBS) of ssODN, 4 ul of Cas9 RNP, and 100 ul of cells were mixed, transferred to a cuvette, and nucleofected according to the manufacturer's instructions (Lonza) using program CA137. The cells were then plated on to one well of a 6-well plate, pre-treated with Matrigel hESC-qualified Matrix (BD) , in TeSR-E8 media containing lOuM ROCK inhibitor (Y-27632, dihydrochloride monohydrate ; Sigma) . The following day the media was changed to TeSR-E8 (minus ROCK inhibitor) and 48 hours after nucleofection the cells were harvested for subcloning and archiving. For some iPS cell lines, particularly early passage iPS cells, pre- treatment of the culture with ROCK inhibitor prior to harvesting the cells for nucleofection may be important to ensure good cell survival .

Subcloning and archiving hiPS clones

Two to four days after nucleofection the well of the 6-well plate was treated with Accutase for 10 min at 37°C to obtain a single cell suspension. Cells were counted and 1,000 or 2,000 cells were plated on 10cm plates, pre-treated with Synthemax II-SC Substrate, in TeSR- E8 containing lOuM ROCK inhibitor. The remaining cells were centrifuged and frozen in 1ml Knockout Serum Replacement (KSR, LifeTech) containing 10% DMSO. The tubes were placed at -80°C overnight prior to long term storage in liquid nitrogen.

The next day, the media on the 10 cm plates was changed to TeSR-E8 (minus ROCK inhibitor) and the cells were grown for around 10 days until good size colonies appeared. Colonies were picked in 96 well plates as follows. The 10 cm plates were washed once with PBS and treated with Accutase for 3-4 minutes at 37°C. The Accutase was replaced with 10 ml of TeSR-E8 media and colonies were picked in 50ul media using a Gilson pipette and transferred into a well of a round bottom 96 well plate containing 50 ul of TeSR-E8 with 20uM ROCK inhibitor. Once all clones were picked the colonies were triturated extensively with a multichannel pipette and the cell suspension was replica plated onto two flat bottom 96 well plates, pre-treated with Matrigel hESC-qualified Matrix, containing 150ul TeSR-E8 with lOuM ROCK inhibitor. The next day, the media was changed to TeSR-E8 (minus ROCK inhibitor) and the cells were grown until most of the wells reached 80% confluence.

To archive the 96-well plate of clones, each well was washed once with 200ul PBS. 30 ul Accutase was then added to each well and the plate was incubated at 37°C for up to 10 minutes. 70ul of KSR was added with a multichannel pipette and single cell suspensions were obtained by trituration. The cells in a total volume of lOOul were then transferred into 96 well Matrix 0.5ml 2D tubes (Thermo Scientific) containing lOOul of KSR+20% DMSO. The tubes were then overlain with 150ul of mineral oil (Sigma) and placed at -80°C overnight prior to long term storage in liquid nitrogen.

Genotyping of iPS cell clones

To genotype the clones, each well of the second 96-well replica plate was lysed with lOOul of gentle lysis buffer (1-1.5 ug/ml proteinase K powder freshly prepared in 50mM KC1, lOmM TrisHCl pH8.3, 2mM MgC12, 0.45% NP40, 0.45% Tween20 buffer) and incubated for at least 4 hours at 55°C. The samples were incubated at 95°C for 10 minutes to inactivate proteinase K and the lysates were diluted 1 in 10 with lOmM Tris HC1 pH8. 2 ul of the diluted lysates were used for PCR amplification with LongAmp Taq DNA Polymerase (NEB) according to the manufacturer's instructions. PCR products were treated with exonuclease and alkaline phosphatase (NEB) and diluted 1 in 4 before Sanger sequencing. REFERENCES

1. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5; 157 (6) : 1262-78. doi: 10.1016/j . cell .2014.05.010.

2. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15 ; 339 ( 6121 ) : 819-23. doi:

10.1126/science.1231143. Epub 2013 Jan 3.

3. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9.

Science. 2013 Feb 15 ; 339 ( 6121 ): 823-6. doi: 10.1126/science .1232033. Epub 2013 Jan 3.

4. Jasin M. Genetic manipulation of genomes with rare-cutting endonucleases . Trends Genet. 1996 Jun; 12 ( 6) : 224-8.

5. Bibikova M, Golic M, Golic KG, Carroll D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.

Genetics. 2002 Jul; 161 (3) : 1169-75.

6. Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG 2nd, Tan W, Penheiter SG, Ma AC, Leung AY, Fahrenkrug SC, Carlson DF, Voytas DF, Clark KJ, Essner JJ, Ekker SC. In vivo genome editing using a high-efficiency TALEN system. Nature. 2012 Nov

1;491 (7422) :114-8. doi: 10.1038 /naturell537. Epub 2012 Sep 23.

7. Yang L, Guell M, Byrne S, Yang JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang PY, Daley G, Church G.

Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 2013 Oct; 41 (19) : 9049-61. doi: 10.1093/nar/gkt555. Epub 2013 Jul 31. 8. Miyaoka Y, Chan AH, Judge LM, Yoo J, Huang M, Nguyen TD,

Lizarraga PP, So PL, Conklin BR. Isolation of single-base genome- edited human iPS cells without antibiotic selection. Nat Methods. 2014 Mar; 11 (3) : 291-3. doi : 10.1038/nmeth.2840. Epub 2014 Feb 9.

9. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC . Highly efficient endogenous human gene correction using designed zinc-finger

nucleases. Nature 2005 435: 646-651. doi : 10.1038/nature03556.

PubMed: 15806097. 10. Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, aite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S . 2008 Apr 15; 105 (15) : 5809-14. doi: 10.1073/pnas .0800940105. Epub 2008 Mar 21.

11. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Mora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol . 2009 Sep; 27 ( 9) : 851-7. doi: 10.1038/nbt.l562. Epub 2009 Aug 13.

12. Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordonez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ, Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A, Vallier L. Targeted gene correction of al-antitrypsin deficiency in induced pluripotent stem cells. Nature. 2011 Oct 12; 478 (7369) :391-4. doi: 10.1038/naturel042 . 13. Chen F, Pruett-Miller SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frodin M, Davis GD. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat

Methods. 2011 Jul 17 ; 8 ( 9) : 753-5. doi: 10.1038/nmeth .1653. 14. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011 Jul 7 ; 29 ( 8 ) : 731-4. doi : 10.1038/nbt .1927.

15. Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K,

Kuperwasser N, Motola DL, Meissner TB, Hendriks WT, Trevisan ,

Gupta RM, Moisan A, Banks E, Friesen M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa E, Wann A, Ahfeldt T, Daheron L, Zhang F, Rubin LL, Peng LF, Chung RT, Musunuru K, Cowan CA. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell. 2013 Feb 7; 12 (2) : 238-51. doi: 10.1016/j . stem.2012.11.011. Epub 2012 Dec 13.

16. Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K.

Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 2013 Apr 4;12 (4) :393-4. doi: 10.1016/j . stem.2013.03.006.

17. Soldner F, Laganiere J, Cheng AW, Hockemeyer D, Gao Q, Alagappan R, Khurana V, Golbe LI, Myers RH, Lindquist S, Zhang L, Guschin D, Fong LK, Vu BJ, Meng X, Urnov FD, Rebar EJ, Gregory PD, Zhang HS,

Jaenisch R. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell. 2011 Jul 22;146 (2) :318-31. doi: 10.1016/j . cell .2011.06.019. Epub 2011 Jul 14.

18. Reinhardt P, Schmid B, Burbulla LF, Schondorf DC, Wagner L, Glatza M, Hoing S, Hargus G, Heck SA, Dhingra A, Wu G, Muller S, Brockmann K, Kluba T, Maisel M, Kruger R, Berg D, Tsytsyura Y, Thiel CS, Psathaki OE, Klingauf J, Kuhlmann T, Klewin M, Muller H, Gasser T, Scholer HR, Sterneckert J. Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell. 2013 Mar 7 ; 12 ( 3 ) : 354-67. doi: 10.1016/j . stem.2013.01.008. 19. Bellin M, Casini S, Davis RP, D'Aniello C, Haas J, Ward-van

Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL . Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. EMBO J. 2013 Dec 11; 32 (24) : 3161-75. doi: 10.1038/emboj .2013.240. Epub 2013 Nov 8. 20. Ochiai Hi, Miyamoto T, Kanai A, Hosoba K, Sakuma T, Kudo Y, Asami K, Ogawa A, Watanabe A, Kajii T, Yamamoto T, Matsuura S.

TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUBlB as causative of PCS (MVA) syndrome. Proc Natl Acad Sci U S A. 2014 Jan 28; 111 (4) : 1461-6. doi: 10.1073/pnas.1317008111. Epub 2013 Dec 16.

21. Bell CC, Magor GW, Gillinder KR, Perkins AC. A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing. BMC Genomics. 2014 Nov 20; 15: 1002. doi: 10.1186/1471-2164-15-1002

22. Kim S, Kim D, Cho SW, Kim J, Kim JS . Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9

ribonucleoproteins . Genome Res. 2014 Jun; 24 ( 6) : 1012-9. doi:

10.1101/gr.171322.113. Epub 2014 Apr 2. PMID: 24696461

23. Lin S, Staahl B, Alia RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014 Dec 15;3. doi: 10.7554/eLife .04766. PMID:

25497837

24. Shah SA, Erdmann S, Mojica FJ, Garrett RA. Protospacer

recognition motifs: mixed identities and functional diversity.

RNA Biol. 2013 May;10 (5) :891-9. doi: 10.4161/rna.23764.

25. Nishimasu Hi, Ran FA2, Hsu PD2, Konermann S3, Shehata SI3, Dohmae N4, Ishitani R5, Zhang F6, Nureki 07. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb

27; 156 (5) : 935-49. doi: 10.1016/j . cell .201 .02.001. Epub 2014 Feb 13. 26. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013 Mar; 31 (3) : 233-9. doi : 10.1038/nbt .2508.

Claims

1. A method for generating a specific biallelic genetic

modification in the genome of a cell and screening for cells that comprise said biallelic genetic modification, the method comprising:

(a) introducing a first RNA-guided endonuclease, a first guide

RNA (gRNA) and a first single-stranded DNA oligonucleotide

(b) culturing the cell under conditions that allow binding of the first RNA-guided endonuclease and gRNA to the target DNA sequence as a first RNA-guided ribonucleoprotein (RNP) complex, cleavage of the target DNA sequence by the first RNA-guided

ribonucleoprotein complex to produce a specific double-stranded break, and homology-driven repair (HDR) between the target DNA sequence and the first ssODN;

(c) obtaining DNA from the cell;

(f) identifying a cell which gives a clean readable sequence trace; and

(g) selecting a cell which is homozygous for the at least one nucleotide modification.

2. The method according to claim 1, wherein the first RNA-guided endonuclease is Cas9.

3. The method according to claim 1 or 2, wherein the first gRNA is a dual guide RNA.

4. The method according to claim 1 or 2, wherein the first gRNA is a single guide RNA.

5. The method according to claim 1 or 2, wherein the cell is a eukaryotic cell.

6. The method according to claim 5, wherein the cell is a mammalian cell or a plant cell.

7. The method according to claim 6, wherein the cell is a stem cell, such as a mammalian embryonic stem cell, a mammalian induced pluripotent stem cell, a mammalian adult stem cell (e.g. a neural stem cell or an organoid stem cell) , a mammalian cancer stem cell, a mammalian immortalised cell line (e.g. a HeLa cell), or a mammalian haploid or near-haploid cell line.

8. The method according to any one of the preceding claims, wherein the at least one nucleotide modification in the first ssODN relative to the target DNA sequence is (a) substitution of at least one nucleotide, (b) deletion of at least one nucleotide, or (c) insertion of at least one nucleotide.

9. The method according to any one of the preceding claims, wherein the at least one nucleotide modification in the first ssODN relative to the target DNA sequence is a single nucleotide

substitution, such that the method generates a single point mutation in the genome of the cell.

10. The method according to any one of claims 1 to 8, wherein there are multiple nucleotide modifications in the first ssODN relative to the target DNA sequence, such that the method generates multiple nucleotide modifications in the genome of the cell.

11. The method according to claim 10, wherein there are multiple nucleotide substitutions in the first ssODN relative to the target DNA sequence, such that the method generates multiple point

mutations in the genome of the cell.

12. The method according to claim 10, wherein there are at least two nucleotide modifications in the first ssODN relative to the target DNA sequence.

13. The method according to any one of claims 10 to 12, wherein the multiple nucleotide modifications are adjacent to each other.

14. The method according to any one of the preceding claims, wherein the at least one nucleotide modification introduces a premature stop codon.

15. The method according to any one of the preceding claims, wherein the at least one nucleotide modification is in an exon.

16. The method according to any one of claims 1 to 14, wherein the at least one nucleotide modification is in a splice site.

17. The method according to any one of claims 1 to 14, wherein the at least one nucleotide modification is in a regulatory sequence, such as a promoter or an enhancer.

18. The method according to any one of the preceding claims, wherein the at least one nucleotide modification is in a non-coding RNA sequence, such as a micro RNA or a long non-coding RNA.

19. The method according to any one of the preceding claims, wherein the method further comprises selecting a cell which is heterozygous for the at least one nucleotide modification.

20. The method according to any one of the preceding claims, wherein following selection of a cell which is homozygous or heterozygous for the at least one nucleotide modification, the method further comprises the following steps:

(h) introducing a second RNA-guided endonuclease, a second gRNA and a second ssODN into the cell, wherein the second gRNA is

complementary to the modified target DNA sequence and wherein the second ssODN is complementary to the modified target DNA sequence but includes at least one nucleotide modification relative to the modified target DNA sequence;

(i) culturing the cell under conditions that allow binding of the second RNA-guided endonuclease and gRNA to the modified target DNA sequence as a second RNA-guided ribonucleoprotein (RNP) complex, cleavage of the modified target DNA sequence by the second RNA- guided RNP to produce a specific double-stranded break, and

homology-driven repair (HDR) between the modified target DNA sequence and the second ssODN;

(j) obtaining DNA from the cell;

(m) identifying a cell which gives a clean readable sequence trace; and

21. The method according to claim 20, wherein the second ssODN is complementary to the wild-type target DNA sequence and step (n) comprises the step of selecting a cell which comprises the wild type target sequence, thereby identifying a cell that has a biallelic revertant phenotype.

22. The method according to any one of claims 1 to 19, wherein the method further comprises the following steps:

(h) introducing a second RNA-guided endonuclease and a second gRNA into a cell which is heterozygous for the at least one nucleotide modification relative to the modified target DNA sequence, wherein the second gRNA is complementary to the modified target DNA sequence ;

(j) obtaining DNA from the cell;

(m) identifying a cell which gives a clean readable sequence trace; and

(n) selecting a cell which is homozygous for the wild type target DNA sequence.

23. The method according to claim 21 or 22, wherein the cell selected in step (n) is tested for function of the wild type target DNA sequence to confirm that function has been restored in the cell.

24. The method according to any one of claims 20 to 23, wherein the second RNA-guided endonuclease is Cas9.

25. The method according to any one of claims 20, 21, 23 or 24, wherein the method further comprises selecting a cell which is heterozygous for the at least one nucleotide modification relative to the modified target DNA sequence.

26. The method according to any one of claims 1 to 21, 23 or 24, wherein the method comprises introducing at least two first gRNAs and at least two first ssODNs into the cell, such that at least two target DNA sequences are modified by the method.

27. A cell produced by the method of any one of the preceding claims .

28. A kit of parts for carrying out the method of any one of claims 1 to 26.

29. A kit according to claim 28, wherein the kit comprises (i) an RNA-guided endonuclease, (ii) a gRNA, (iii) a first ssODN, (iv) a first PCR primer, and (v) a second PCR primer, wherein the first gRNA is complementary to a target DNA sequence to be modified in the genome of a cell, the first ssODN is also complementary to the target DNA sequence, but includes at least one nucleotide

modification relative to the target DNA sequence, the first PCR primer binds upstream of the target DNA sequence and the second primer binds downstream of the target DNA sequence.