WO2020036653A2 - Improved method for homology directed repair in cells - Google Patents

Abstract

A method to increase the efficiency of homology-directed repair (HDR) is provided. For example, the method employs of a chromatin HDR donor template instead of a plain ("naked") DNA donor template and may be used for the insertion of DNA sequences, such as a gene, at specific genomic locations, but it can be used for insertion, deletion, or modification of DNA sequences. The method can be used in conjunction with any method for the generation of specific double-strand DNA breaks (DSBs) or with other methods for the enhancement of the efficiency of HDR in cells.

Description

IMPROVED METHOD FOR HOMOLOGY DIRECTED REPAIR

IN CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. application No. 62/662,655, filed on April 25, 2018, the disclosure of which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grant number GM118060 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

For genome editing in cells, the state-of-the-art technology involves the use of DNA sequence-specific nucleases that generate double-strand DNA breaks (DSBs) at specifically targeted sites in the genome (for reviews, see: Carroll, 2014; Harrison et al., 2014). The three major types of sequence-specific nucleases used in genome engineering are: (i) zinc finger nucleases (ZFNs); (ii) transcriptional activator-like effector nucleases (TALENs); and (iii) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 RNA-guided nucleases (RGNs).

At present, CRISPR-Cas9 and related RGNs are the most widely used sequence-specific nucleases. RGNs or RNA-guided nucleases are

ribonucleoprotein complexes whose sequence-specific guide RNA (gRNA) moieties recruit a nuclease such as Cas9 to a DNA target site. Base pairing between the gRNA and sequences in the target DNA triggers Cas9-mediated double-stranded DNA break (DSB) formation. The repair of RGN-induced targeted DNA lesions can occur by non-homologous end joining (NHEJ) or by homologous recombination (via homology directed repair, HDR) to result in the deletion, addition, or modification of genetic information in cells from almost any organism.

DSBs are generally repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). In NHEJ, the two flee ends of the DSB are ligated together, usually imperfectly with small DNA deletions or insertions. In normal HDR in cells, a homologous DNA sequence from a sister chromosome is used as a donor template to repair the damaged chromosome containing the DSB. In genome engineering, the repair of DSBs by HDR involves the addition of a customized donor DNA template. The customized donor DNA template is typically designed in a manner that leads to the modification of specific nucleotides or the insertion of a DNA sequence, such as a gene, at a specific genomic location, e.g., the introduction of specifically desired sequences or insertions in the region of the DSB.

For many applications, it is essential to use HDR instead of NHEJ because HDR mediates the modification or insertion of customized DNA sequences at a specific genomic location rather than simple gene disruption, as would generally occur with NHEJ. There is, however, a competition between the use of NHEJ and HDR for the repair of DSBs. Notably, HDR occurs much less efficiently than NHEJ.

There has been considerable effort devoted to increasing the efficiency of HDR. Some examples are as follows. To promote HDR relative to NHEJ, the efficiency of NHEJ was reduced by using mutant cells that lack DNA ligase IV, an important enzyme in the NHEJ pathway. The loss of DNA ligase IV resulted in a substantial increase in HDR relative to NHEJ in Drosophila (Beumer et al., 2008; Bozas et al., 2009). However, for most applications, it is not possible to knock out the gene encoding DNA ligase IV.

By drag-induced cell cycle synchronization, Lin et al. (2014) and Yang et al. (2016) were able to achieve the enhancement (1.6- to 3.3-fold and 3- to 6- fold, respectively) of HDR in mammalian cells.

By reducing Cas9 expression in G1 phase and increasing Cas9 expression in S, G2, and M phases of the cell cycle, Gutschner et al. (2016) were able to achieve up to a 1.87-fold increase in the efficiency of HDR

Guo et al. (2018) found that cold shocking iPS cells at 32°C increased HDR by 1.8- to 3 6-fold. When the basal level of HDR was very low (<0.2%), they could observe about a 10-fold increase in HDR efficiency in HEK293 cells by cold shock treatment. The use of small molecules (such as NU7441 and Ku-0060648) that inhibit DNA-PK resulted in 2- to 4-fold enhancement of HDR in mammalian cells (Robert et al., 2015).

Finder et al. (2015) showed a 3- to 6-fold enhancement of HDR in mammalian cells through the overexpression or the stimulation of Rad51 by the small molecule RSI.

Takayama et al. (2017) were able to achieve higher levels of biallelic gene editing in ES/iPS cells by a combination of Rad51 overexpression and treatment of valproic acid.

Charpentier and colleagues (2018) showed a 2-fold increase in HDR by- using a Cas9 protein that is fused to a fragment of CtIP, which is a key protein in the early steps of homologous recombination.

The microinjection of a combination of Cas9 protein, multiple RNA species, and long single-stranded DNA into mouse embryos (~20 to 105 zygotes injected per experiment) resulted in a moderate level of success in the achievement of heterozygous (and much less commonly, homozygous) directed insertions in the resulting live pups (1 to 6 per experiment) (Quadras et al., 2017).

Thus, the most common gene editing technique is based on the CRISPR- Cas9 system, and the most common way of performing gene editing is by adding the donor DNA template as circular DNA, linear DNA, or ssDNA (single stranded DNA).

SUMMARY

Herein, it is shown that the efficiency of HDR in cells is increased by the use of an exogenously supplied chromatin donor template rather than a naked DNA donor template, e.g., increased in a manner that does not require the use of mutant cells, drug treatment, cold shock, a drug regulated cell cycle, or expression or addition of an exogenous protein that is not a nuclease. The success of the method disclosed herein is likely to be due to the use of the more natural form of the donor template as chromatin rather than as unnatural naked DNA. Chromatin is a complex of DNA and proteins termed the core histones. In chromatin, the DNA and histones are organized into particles known as nucleosomes. The nucleosome is the unit repeat of chromatin and is composed of about 180 to 200 bp of DNA and two copies each of the core histones H2A, H2B, H3, and H4. When examined by electron microscopy, chromatin has a "beads-on-a-string" appearance, where each of the "beads" is a nucleosome.

Thus, the disclosure provides a method for increasing the efficiency of homology directed repair (HDR) in cells by using a chromatin donor template. High cell death rates are observed in transfected cells undergoing gene editing with a naked DNA donor template; however, the disclosed method reduces cell death considerably and increases HDR efficiency by, for example,

2- to 7-fold, as shown by the positive effect of chromatin donor templates (as compared with naked DNA templates) in genome editing , e.g., HDR-CRISPR- mediated genome editing, e.g., monitored by the site-directed insertion of a reporter gene in human MCF10A or human HeLa cells. In other words, the disclosed method is a performance improvement on existing gene editing methods. Thus, the use of DNA donor templates assembled into chromatin enhances the rate of HDR for precise genome editing (for example when using the CRISPR-Cas9 system) and increases cell survival.

In one embodiment, the method is employed to introduce a new sequence or gene (“donor template”) as chromatin into a host chromosome via gene editing techniques, e.g., CRISPR-Cas9, though other nucleases including but not limited to Casl2a, CasX, CasY or Cpfl may be used in lieu of Cas9, as well as Zinc finger nucleases (ZFNs) or transcriptional activator-like effector nucleases (TALENs). In one embodiment, the method may be employed to correct a gene defect in a cell. In one embodiment, the method may be employed to treat patients suffering various diseases via gene therapy. In one embodiment, the method may be employed to change a biological function or process in a plant, animal, human, etc.

The method may thus be employed in any gene editing system, e.g., a CRISPR gene editing system, for uses including gene therapy, agriculture or any other field where introduction of a new sequence or gene, or modification of a gene, would be useful. The method increases the efficiency of homology directed repair and/or reduces cell death, e.g., relative to methods that do not employ chromatin as a donor template. In one embodiment, the donor template has coding sequences, e.g., one or more exons or portions thereof, optionally including one or more introns or portions thereof. In another embodiment, the donor template has a transcriptional regulatory sequence, e.g., a promoter, enhancer, insulator/boundary element, transcriptional termination sequence, or intron. In addition to the insertion of sequences, the method may be used to remove undesired sequences, such as repetitive DNA sequences that occur in many disease-associated genes, such as the human huntingtin ( HΊT) gene (Huntington's Disease; HD) or the human frataxin (FXN) gene (Friedreich's Ataxia; FRDA).

In one embodiment, a method to enhance the efficiency of homology directed repair in mammalian cells is provided. The method includes contacting the cells with an effective amount of chromatin (histones complexed with DNA to be employed in the homology directed repair) and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease, under conditions allowing for enhanced homology directed repair in one or more chromosomes of the mammalian cells relative to mammalian cells contacted with isolated nucleic acid that is not complexed as chromatin (that is not in a chromatin complex) to be employed in the homology directed repair and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease. In one embodiment the DNA is mammalain DNA. Chromatin employed in the method is prepared, in one embodiment, by combining isolated histones and isolated nucleic acid to be employed in the homology directed repair (the“donor template”). The donor template includes homology arms flanking a sequence of interest, e.g., a sequence to be inserted into a chromosome of the cell and expressed in the cell, a sequence to replace a sequence in the chromosome of the cell, or a sequence that allows for a deletion of a sequence in the chromosome of the cell. In one embodiment, the nucleic acid complexed as chromatin encodes a protein. In one embodiment, the nuclease comprises a Cas protein, a ZFN or a TALEN. In one embodiment, the cells are in a mammal. In one embodiment, the mammal is a human. In one embodiment, the mammal is a bovine, caprine, ovine, swine, canine, feline, or non-human primate. In one embodiment, the repair results in an insertion. In one embodiment, the repair results in a deletion. In one

embodiment, the repair results in replacement of a sequence in the cells. In one embodiment, the repair results in an insertion of a sequence in the cells. In one embodiment, the nuclease is encoded on a plasmid that is contacted with the cell. In one embodiment, further comprises guide RNA. In one embodiment, the cells are contacted with a composition comprising chromatin and the nuclease. In one embodiment, the cells are contacted with a composition comprising chromatin containing nucleic acid to be employed in the homology directed repair and isolated nucleic acid encoding the nuclease. In one embodiment, the length of the nucleic acid to be employed in the homology directed repair is at least 150 bp. In one embodiment, the length of the nucleic acid to be employed in the homology directed repair is at least 3000 bp. In one embodiment, the length of the nucleic acid to be employed in the homology directed repair is less than about 10 kbp. In one embodiment, the enhancement is at least 2-fold. In one embodiment, about 150 to 200 bp of nucleic acid to be employed in the homology directed repair is complexed with 2 molecules of each of the core histones H2A, H2B, H3, and H4. In one embodiment, the method results in biallelic homology directed repair.

Further provided is an in vivo method to enhance the efficiency of homology directed repair in a plant or animal, comprising: contacting cells of the plant or administering to the animal an effective amount of a composition comprising chromatin comprising isolated nucleic acid to be employed in the homology directed repair and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease. In one embodiment, the administration is local. In one embodiment, the administration is systemic. In one embodiment, the nucleic acid to be employed in the homology directed repair has at least one nucleotide substitution, insertion or deletion relative to nucleic acid in the cells of the plant or animal .

BRIEF DESCRIPTION OF FIGURES

Figures 1A-C. Schematic representation of the CRISPR-Cas9 target regions for HDR-mediated insertion of a GFP reporter sequence. (A) GAPDH locus. The T2A-GFP sequence was inserted close to the 3' untranslated region in exon 9 (E9) of the GAPDH locus. The 5' and 3' homology arms (HA; ~0.9 kb each) and GFP sequence were subcloned into the pBluescript KS vector and used as the HDR donor template. (B) RAB11A locus. The GFP sequence was inserted in the first exon (El) of the RAB11A locus. The in-frame HDR-mediated insertion of GFP into the RAB11A coding sequence produces an N-terminally- tagged RAB 11 A protein. The HDR donor template comprises homology arms (~1.1 kb each) flanking the GFP sequence.

(C) ACTB locus. The mEGFP sequence was inserted into the second exon (E2) of the ACTB locus. The in-frame HDR-mediated insertion of mEGFP in the

ACTB coding sequence produces an N -terminally-tagged ACTB protein. The HDR donor template comprises homology arms (~1 kb each) flanking the mEGFP sequence. The dashed lines indicate the regions of homology between the HDR donor templates and the CRISPR-Cas9 target loci. The black boxes represent coding regions, and white boxes represent untranslated regions. E, exon; HA, homology arm.

Figures 2A-D. Strategy for testing the effects of chromatin versus naked DNA donor templates in CRISPR-Cas9-mediated HDR (A) Diagram of plasmids used in the CRISPR-Cas9-mediated gene editing experiments. The Cas9-T2A-mCherry sequence and the target-specific single guide RNA (sgRNA) sequence were included in the same plasmid. The HDR donor template plasmids contained the GFP (or mEGFP) sequence flanked by two target- specific homology arms. The HDR donor templates were used as either naked DNA or chromatin during cell transfection. (B) Salt dialysis reconstitution of chromatin. The HDR donor template plasmids were reconstituted into chromatin with purified core histones by the salt dialysis method. (C)

Micrococcal nuclease digestion analysis of chromatin reconstituted with purified components. Preparations of chromatin that were reconstituted with each of the HDR donor template plasmids (which correspond to the GAPDH, RAB 11 A, and ACTB loci, as in Fig. 1) were subjected to partial digestion with four different concentrations of micrococcal nuclease. The samples were deproteinized, and the resulting DNA fragments were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide. The arrows indicate the DNA bands that correspond to mono-, di-, tri-, tetra-, and pentanucleosomes. The DNA size markers (M) are the 123-bp ladder (Millipore Sigma). (D) Schematic outline of the workflow in the CRISPR-Cas9-mediated editing experiments with chromatin or naked DNA donor templates. Plasmid DNA containing the coding sequence for Cas9-T2A-mCherry and a target-specific sgRNA sequence was cotransfected into human cells (MCF10A or HeLa cells) with the corresponding HDR donor template as naked DNA or chromatin. At 24 hours post- transfection, cells that express Cas9-T2A-mCherry were enriched by FACS and cultured for an additional 10 days. The expression of GFP was analyzed by flow cytometry, and individual GFP-positive cells were sorted by FACS to generate independent clones. To determine whether there was partial or complete conversion of the multiple chromosomes containing the target genes, the GFP insertions in each of the independent clones was analyzed by PCR These experiments were performed under standard CRISPR-Cas9 genome editing conditions, such as those described by Ran et al. (2013), except that we used chromatin HDR donor templates instead of naked DNA HDR donor templates.

FACS, fluorescence-activated cell sorting; PCR, polymerase chain reaction.

Figures 3A-C. The efficiency of HDR-mediated gene editing with CRISPR-Cas9 is higher with chromatin donor templates than with naked DNA donor templates. (A) Flow cytometry' analysis reveals an increase in the incorporation of GFP coding sequence with chromatin relative to DNA donor templates. HDR experiments were performed as outiined in Fig. 2D with human MCF10A cells and the three donor templates ( GAPDH , RAB11A and ACTB) shown in Fig. 1. The percentage of GFP-positive cells (out of a total of 200,000 cells analyzed per experimental condition) is indicated in each plot. The population of GFP-positive cells was gated based on control cells (untransfected) that show no GFP expression. These data represent one out of three independent experiments. (B) Combined results from three independent experiments (n = 3) performed as in Fig. 3A. The average and standard deviation are indicated for each set of experiments. (C) The use of chromatin relative to naked DNA donor templates results in a 2.3- to 6.8-fbld enhancement of HDR. The graph shows the fold enhancement of HDR with chromatin relative to naked DNA donor templates and is based on data from Fig. 3B.

Figures 4A-C. The use of chromatin HDR donor templates results in an increase in the efficiency of HDR-mediated biallelic gene editing relative to that seen with naked DNA donor templates. (A) PCR analysis of edited genomic DNA. HDR experiments were performed as shown in Fig. 2D, and the genomic DNA from individual GFP-positive clones were analyzed by PCR. The positions of the PCR amplification products from edited and wild-type chromosomes are shown. The PCR products derived from untreated wild-type cells are also included. The asterisks indicate imperfect clones that appear to lack a properly edited chromosome, as indicated by either the absence of an edited chromosome or the presence of a PCR product whose size is not consistent with that of an edited or wild-type chromosome. This figure depicts the results from a representative subset of the GFP-positive clones. (B) Summary of the PCR analysis of clones obtained in the HDR-mediated insertion of GFP at the GAPDH, RAB11A, and ACTB loci. MCF10A cells are diploid, and thus have two copies of each of these loci. The clones were classified as biallelic (with two edited chromosomes), monoallelic (with one edited chromosome and one wild-type chromosome), or imperfect, as defined in Fig. 4A. (C) The percentages of clones that correspond to the categories of data presented in Fig.

4B.

Figures 5A-E. The efficiency of HDR-mediated gene editing with CRISPR-Cas9 is higher with chromatin donor templates than with naked DNA donor templates in HeLa cells. (A) Flow cytometry' analysis shows an increase in the incorporation of GFP coding sequences with chromatin relative to naked DNA donor templates. Experiments were performed as in Fig. 3 with the GAPDH locus in HeLa cells, except that the population of mCherry-positive cells was not enriched. (B) The use of chromatin relative to naked DNA donor templates results in a 2-fold enhancement of HDR. The graph is based on data from Fig. 5A. (C) The use of chromatin HDR donor templates results in an increase in the efficiency of homozygous edited clones relative to that seen with naked DNA donor templates. PCR analysis of edited genomic DNA was carried out as in Fig. 4A. The positions of the PCR amplification products from edited and wild-type chromosomes are shown. The PCR products from untreated wild- type cells are also included. HeLa cells have four copies of the chromosome containing the GAPDH gene. This figure depicts the results from a subset of the GFP-positive clones. (D) Summary of the PCR analysis of clones obtained in the HDR-mediated insertion of GFP at the GAPDH locus in HeLa cells. The homozygous clones have four copies of the integrated GFP sequence; the heterozygous clones have one to three copies of the integrated GFP sequence; and the imperfect clones appear to lack a properly edited chromosome, as indicated by either the absence of an edited chromosome or the presence of a PCR product whose size is not consistent with that of an edited or wild-type chromosome. (E) The percentages of clones that correspond to the categories of data shown in Fig. 5D.

Figures 6A-B. Transfection of cells with chromatin is less toxic than transfection of cells with naked DNA. (A) Transfection of MCF10A cells. The cell viability after transfection with the GAPDH HDR donor construct as either naked DNA or chromatin was determined along with the viability of mock (no DNA or chromatin) transfected cells. The analysis was performed 24 h after transfection. The cell viability was assessed by flow cytometry in the presence of DAPI. The average and standard deviation from three independent experiments are shown. (B) Transfection of HeLa cells. The cell viability after transfection with the GAPDH HDR donor construct as either naked DNA or chromatin was determined along with the viability of mock (no DNA or chromatin) transfected cells. The analysis was performed 3 days after transfection. The cell viability' was assessed by flow cytometry in the presence of DAPI (4',6-diamidino-2-phenylindole). Live cells are impermeant to DAPI. The average and standard deviation from three independent experiments are shown.

DETAILED DESCMPTION

The disclosure provides a method for increasing the efficiency of homology-directed repair (HDR) in cells by the use of a chromatin HDR donor template instead of a plain ("naked") DNA donor template. This method is particularly useful for the insertion or modification of DNA sequences, such as a gene, at specific genomic locations. This technology can be used in conjunction with any method for the generation of specific double-strand DNA breaks and alteration of genomic DNA . It was found that transfection with chromatin is less toxic to cells than transfection with naked DNA. Therefore, the use of chromatin as a donor template for HDR experiments increases the efficiency of HDR as well as the viability of the cells relative to the use of naked DNA. Moreover, a chromatin HDR donor template can be used in conjunction with other methods for the enhancement of the efficiency of HDR in cells, e.g., other methods disclosed herein. There is considerable potential for the use of HDR in the treatment of human genetic diseases, which includes some forms of cancer (see, for example, Sanchez-Rivera and Jacks, 2015). The disclosed method would increase the rate of success of such treatments. Moreover, the method could be used in conjunction with other methods to increase the efficiency of HDR as well as to minimize cell death during the HDR process. In many instances, the use of the disclosed method may be the difference between success and failure in treatments. In addition, the method allows for enhanced efficiencies in performing genetic engineering on cells or organisms to obtain pharmaceuticals, crops, biofuels, etc.

The disclosed method results in enhanced efficiencies, e.g., relative to naked DNA based methods including but not limited to the following.

The method of Canny et al. (2018) used circular (naked DNA) HDR donor templates and Cas9 ribonucleoprotein complexes, in combination with an inhibitor of 53BP1. 53BP1 is a key regulator of DSB repair that promotes the occurence of NHEJ over HDR. Thus, the inhibition of 53BP1 decreases the occurrence of NHEJ relative to HDR. The method disclosed herein may be employed with an inhibitor of 53BP1.

Chu et al. (2015) described methods that use plasmid encoded Cas9 and linear or circular HDR (naked DNA) donor templates in combination with different strategies to suppress NHEJ. In this manner, they were able to achieve up to an eight-fold increase in the efficiency of HDR. The method disclosed herein may be employed with different strategies to suppress NHEJ disclosed in Chu et al. (2015).

In the method developed by Roth et al. (2018), purified components (Cas9 protein and sgRNA) are assembled into ribonucleoprotein complexes and combined with PCR-amplified naked DNA donor repair templates during cell transfections. Thus, in these experiments, the Cas9 and sgRNA are introduced into the cells as a nucleoprotein complex instead of as DNA constructs. The method disclosed herein may be employed with Cas9 and sgRNA are introduced into the cells as a nucleoprotein complex or as DNA constructs.

The method of Kato-Inui et al. (2018) used circular (naked DNA) HDR donor templates in combination with plasmids encoding high fidelity Cas9 variants to enhance the efficiency of HDR relative to NHEJ. The method disclosed herein may be employed with high fidelity' Cas9 variants. The method of Zhang et al. (2017) used circular (naked DNA) HDR donor templates in which the 5' and 3' homology arm sequences are distally flanked by sgRNA sequences that promote the generation of double cuts in the HDR donor template when combined with Cas9 and the corresponding sgRNA sequence. The method disclosed herein may be employed with donor templates in which the 5' and 3' homology arm sequences are distally flanked by sgRNA sequences.

The method of Li et al. (2018) used a plasmid to express Cas9 and circular HDR donor templates in transfection-sensitive cells that transiently overexpress the BCL-XL protein (an antiapoptotic factor). The method disclosed herein may be employed with an antiapoptotic factor.

The use of chromatin as the donor template in HDR experiments may be combined with any method that uses a donor template that consists of Unear or circular double-stranded DNA that is at least about 150 bp (e.g., corresponding to the length of DNA in a single nucleosome). There is no restriction in the maximum length of DNA that can be assembled into chromatin, e.g., as a donor

DNA template. Some examples of other methods that may be enhanced by the use of a chromatin donor template are as follows.

1. Methods that modulate the expression of the Cas9 nuclease during the cell cycle. The method described by Gutschner et al. (2016) uses circular naked DNA as HDR donor template in combination with a plasmid that encodes a fusion protein that consists of Cas9 and the N-terminal region of Geminin degron, which induces the degradation of Cas9 during the Gl phase of the cell cycle. Because DSB repair occurs only by NHEJ in Gl phase, this method reduces the relative amount of NHEJ relative to HDR A chromatin donor template may be employed instead of a naked DNA donor template for HDR in the method described by Gutschner et al. (2016).

2. Methods that alter the expression level or the activity of proteins involved in the NHEJ or HDR pathways by the use of chemicals or genetic modifications. For example, Finder et al. (2015) uses plasmid encoded Cas9 and circular naked DNA donor templates in combination with the stimulation of Rad51 by the smaU molecule RSI. With this method, a chromatin donor template could be used instead of a naked DNA donor template for HDR. Also, to increase the efficiency of HDR, a chromatin donor template may be employed in the method of Takayama et al. (2017), rather than a plasmid (naked DNA) HDR donor template, in combination with the overexpression of RadSl and valproic acid treatment of cells. Further, a chromatin donor template could be used instead of a naked DNA donor template in the method of Canny et al. (2018) along with Cas9 ribonucleoprotein complexes and an inhibitor of 53BP1. 53BP 1 is a key regulator of DSB repair that promotes the occurence of NHEJ over HDR Thus, the inhibition of 53BP1 decreases the occurrence of NHEJ relative to HDR. A chromatin donor template could be used instead of a naked DNA donor template in the method of Chu et al. (2015) along with plasmid encoded Cas9 in combination with different strategies to suppress NHEJ.

3. Methods that modulate the recruitment of components of the HDR machinery to the site of DSB formation. The method of Chaipentier et al. (2018) uses circular naked DNA as the HDR donor template in conjunction with a fusion protein that contains Cas9 and an N -terminal segment of CtIP, a key protein in the initial step of homologous recombination. The CtIP fragment directs the recruitment of the Cas9-CtIP fusion protein to the site of DBS formation. With this method, a chromatin donor template could be used instead of a naked DNA donor template for HDR.

4. Methods that use HDR donor templates in combination with CRISPR- Cas9 ribonucleoprotein complexes during cell transfection. In the method developed by Roth et al. (2018), purified components (Cas9 protein and sgRNA) are assembled into ribonucleoprotein complexes and combined with PCR- amplified naked DNA donor repair templates during cell transfections. Thus, in these experiments, the Cas9 and sgRNA are introduced into the cells as a nucleoprotein complex instead of as DNA constructs. With this methodology, a chromatin donor template could be used instead of a naked DNA donor template for HDR

5. Methods that use engineered Cas9 enzymes with improved specificity. The method of Kato-Inui et al. (2018) uses circular (naked DNA) HDR donor templates in combination with plasmids coding high fidelity Cas9 variants to enhance the efficiency of HDR relative to NHEJ. These Cas9 variants could potentially be used with chromatin donor templates.

6. Methods that use double cut DNA donor repair templates. The method of Zhang et al. (2017) describes the use of circular (naked DNA) HDR donor templates in which the 5' and 3' homology arm sequences are distally flanked by sgRNA sequences that promote the generation of double cuts in the HDR donor template when combined with Cas9 and the corresponding sgRNA sequence. This method could potentially be modified so that the HDR donor is chromatin instead of naked DNA.

7. Methods that improve cell survival after transfection. The method of Li et al. (2018) uses a plasmid to express Cas9 and circular HDR donor templates in transfection-sensitive cells that transiently overexpress the BCL-XL protein (an antiapoptotic factor). With this method, a chromatin donor template could be used instead of a naked DNA donor template for HDR

8. Methods that alter the cell cycle to optimize HDR The method of Yang et al. (2016) uses nocodazole and the small molecule ABT-751 to induce cell cycle synchronization in the G2/M phase of the cell cycle. Then, plasmids that express wild-type or variant Cas9 are combined with linear or circular HDR (naked DNA) donor plasmids in cell transfection. This results in a 3- to 6-fold increase in HDR. These methods could potentially be used in conjunction with a chromatin donor template instead of a naked DNA donor template.

For example, a chromatin donor template may be employed with Cas9 ribonucleoprotein complexes or DNA encoding Cas9 and sgRNA, in combination with an inhibitor of 53BPlor with plasmids coding high fidelity Cas9 variants; the chromatin donor template may include.

the 5' and 3' homology arm sequences that are distally flanked by sgRNA sequences that promote the generation of double cuts in the HDR donor template when combined with Cas9 and the corresponding sgRNA sequence.

The chromatin HDR donor template may be linear or circular. The donor template includes homology arms which may be from about 0.15 to about 2 kb in length, e.g., about 1 kb in length. The donor sequence (an insert) itself may be of any length, e.g., from 0.15 up to 50 kb. In one embodiment, the donor template is a plasmid having homology arms from about 500 to 1000 bp flanking an insert. In the examples below, tire donor template is a plasmid having vector sequences of about 3 kb, homology arms of about 1 kb and an insert of about 0.7 kb.

When preparing the chromatin donor templates, in one embodiment, there is an average of about one nucleosome per 160 bp DNA as each nucleosome occupies 147 bp DNA. Therefore, there is an average of 13 bp (160 bp -147 bp) of free/naked DNA per nucleosome. In other words, for every 160 bp of DNA, about 147 bp of DNA is bound to histones in a nucleosome and about 13 bp of DNA is free/naked DNA. In most cells, there is an average of about one nucleosome per 180 to 200 bp. Thus, in other embodiments, there is an average of about one nucleosome per 150, 170, 180, 190, 200, 210, 220, 230, 240, or 250 bp DNA. Histones are not added to excess in the chromatin forming reaction as an insoluble aggregate may be formed.

Exemplary Methods

Thus, the disclosure is concerned with the use of a chromatin donor template for HDR. As discussed above, this technology can be used in conjunction with any method for the generation of site-specific double-strand DNA breaks. In addition, transfection with chromatin is less toxic to cells than transfection with naked DNA. Therefore, the use of chromatin as a donor template for HDR experiments increases the efficiency of HDR as well as the viability of the cells relative to the use of naked DNA. As described herein, the efficiency of HDR was shown to be increased by the use of the DNA donor template as chromatin rather than as naked DNA. These experiments were performed in conjunction with the CRISPR-Cas9 RGN system, and it is likely that similar results would be observed with other sequence-specific nucleases. Additionally, the chromatin donor template is less toxic to cells than the corresponding naked DNA donor template during DNA transfections. Thus, the use of chromatin instead of naked DNA for HDR has two benefits: (i) an increase in the efficiency of HDR, and (ii) an increase in the viability of the transfected cells.

HDR experiments with chromatin donor templates were demonstrated to be highly reproducible for any particular experimental condition. In several different types of experiments with either MCF10A cells or HeLa cells, it was found that the efficiency of HDR increases from about 2-fold to 7-fold by the use of a chromatin donor template relative to a naked DNA donor template. As the overall efficiency of HDR increases, the fold-increase in HDR by chromatin generally decreases. For example, when the efficiency of HDR with naked DNA is high (e.g., 33%), the maximum enhancement by chromatin may be 3-fold (100%/33%) and when the efficiency of HDR with naked DNA is low (e.g., 0.1%), then a small increase in efficiency by chromatin (e.g., to 1.5%) would be a 15-fold increase (1 ,5%/0.1%). Under all conditions tested, there was no less than a 2-fold increase in the efficiency of HDR with chromatin relative to naked DNA donor templates. In addition, the use of a chromatin donor template has consistently been found to have a less deleterious effect on the viability of the transfected cells than the use of the corresponding naked DNA donor template. In atypical experiment with HeLa cells, untransfected cells exhibited about 25% cell death, chromatin transfected cells exhibit about 40% cell death, and naked DNA transfected cells exhibit about 60% cell death. Thus, the method yields higher HDR efficiency and lower cell death as a result of the use of a chromatin donor template rather than a naked DNA donor template. Therefore, the use of a natural chromatin donor template results in a higher efficiency of HDR as well as a higher viability of cells throughout the HDR process. This may be of particular importance in vivo, in primary cells, or cells that may only be obtained only in small numbers. Therefore, the use of the method may be the difference between success and failure of HDR with cells, such as some primary, stem or progenitor cells that can be obtained only in small quantities.

The method, which uses a chromatin donor template rather than a naked DNA donor template, does not involve any special treatment (such as the addition of drugs, or cold shock, or cell cycle synchronization) or genetic modification of the cells. Therefore, this method can be used on all types of cells.

The use of a chromatin donor template instead of a naked DNA donor template was shown in standard CRISPR-Cas9 HDR experiments, such as those described in Ran et al. (2013), to result in an increase in the efficiency of HDR and a decrease in the occurrence of cell death during the transfection process. In these experiments, the homology-directed insertion of a reporter gene was demonstrated in two different human cell lines (MCF10A cells and HeLa cells; three loce were used in MCF10A cells and one locus was used in HeLa cells).

Exemplary Embodiments

A method to enhance the efficiency of homology directed repair in cells is provided. In one embodiment, a composition is provided comprising isolated nucleic acid complexed as chromatin to be employed in the homology directed repair. The composition is contacted with cells. The cells may express or be induced to express a nuclease useful in gene editing or may be contacted with a nuclease useful in gene editing or with isolated nucleic acid encoding the nuclease. The amount of the composition and nuclease expression allows for enhanced homology directed repair in one or more chromosomes of the cells relative to cells contacted with nucleic acid to be employed in the homology directed repair that is not complexed with chromatin and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease, or in cells not induced to express the nuclease. In one embodiment, the nucleic add complexed as chromatin encodes a protein. In one embodiment, the nucleic acid complexed as chromatin comprises a transcription regulatory element. In one embodiment, the nuclease comprises a Cas protein, a ZFN or a TALEN. In one embodiment, the cells are in a mammal. In one embodiment, the mammal is a human. In one embodiment, the mammal is a bovine, caprine, ovine, swine, canine, feline, or non-human primate. In one embodiment, the repair results in an insertion. In one embodiment, the repair results in a deletion. In one embodiment, the repair results in replacement of a sequence in the cells. In one embodiment, the cells are plant cells or animal cells. In one embodiment, the nuclease is encoded on a plasmid that is contacted with the cell. In one embodiment, the plasmid further comprises guide RNA. In one embodiment, the isolated nucleic acid to be employed in the homology directed repair is on a plasmid. In one embodiment, the cells are contacted with a composition comprising nucleic acid complexed as chromatin to be employed in the homology directed repair and the nuclease. In one embodiment, the cells are contacted with a composition comprising nucleic acid complexed as chromatin to be employed in the homology directed repair and isolated nucleic acid encoding the nuclease. In one embodiment, the length of the isolated nucleic acid to be employed in the homology directed repair is at least 150 bp and less than about 50 kbp. In one embodiment, the enhancement is at least 2-fold, 4-fold, 6-fold, 8-fold, 10-fold or more. In one embodiment, 150 to 200 bp of isolated nucleic acid is complexed with 2 molecules of each of the core histones H2A, H2B, H3, and H4. In one embodiment, the histones are mammalian histones, e.g. human histones. In one embodiment, the histones are homologous to the cells. In one embodiment, the histones are from non-human animals. In one embodiment, the histones are recombinant histones. In one embodiment, the histones are native histones. In one embodiment, the homology directed repair is biallelic.

In one embodiment, an in vivo method to enhance the efficiency of homology directed repair in a plant or animal is provided. The method includes contacting cells of the plant or administering to the animal an effective amount of a composition comprising nucleic acid complexed as chromatin to be employed in the homology directed repair and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease. In one embodiment, the administration is local. In one embodiment, the administration is systemic. In one embodiment, the isolated nucleic acid has at least one nucleotide substitution, insertion or deletion relative to nucleic acid in the cells of the plant or animal. In one embodiment, the mammal has Barth syndrome, Duchenne muscular dystrophy, hemophilia, b-Thalassemia, cystic fibrosis, chronic granulomatous disease.

Exemplary Nuclease System: CRISPR-Cas System

The Type II CRISPR is a well characterized system that carries out targeted DNA double-strand breaks in four sequential steps. First, two noncoding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre- crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA :tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double- stranded break within the protospacer.

The requirement of the crRNA-tracrRNA complex can be avoided by use of an engineered "single-guide RNA" (sgRNA) that comprises the hairpin normally formed by the annealing of the crRNA and the tracrRNA (see Jinek, et al. (2012) Science 337:816 and Cong et al. (2013)

Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineered tracrRNA:crRNA fusion, or the sgRNA, guides Cas9 to cleave the target DNA when a double strand RNA:DNA heterodimer forms between tire Cas associated RNAs and the target DNA. This system comprises the Cas9 protein and an engineered sgRNA.

"Cas polypeptide" encompasses a full-length Cas polypeptide, an enzymatically active fiagment of a Cas polypeptide, and enzymatically active derivatives of a Cas polypeptide or fiagment thereof. Suitable derivatives of a Cas polypeptide or a fiagment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fiagment thereof.

Donor Molecules for Homology Directed Repair

An exogenous sequence (also called a "donor sequence" or "donor" or "transgene" or“gene of interest”) can be used, for example for correction of a mutant gene or for increased expression of a wild-type gene. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence where it is placed. A donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology' to sequence in the region of interest.

The donor polynucleotide can be DNA, single-stranded and/or double- stranded and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a Unear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang, et al. (1987); Nehls, et al. (1996).

Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by vimses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor man be inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted (e.g., highly expressed, albumin, AAVS1, HPRT, etc.). However, it will be apparent that the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.

The donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into the GAPDH (glyceraldehyde 3- phosphate dehydrogenase) or other locus such that some (N-terminal and/or C- terminal to the transgene encoding the glycolytic enzyme) or none of the endogenous GAPDH sequences are expressed, for example as a fusion with the transgene encoding the enzyme sequences. In other embodiments, the transgene (e.g., with or without additional coding sequences such as for GAPDH) is integrated into any endogenous locus, for example a safe-harbor locus. See, e.g., U.S. Patent Publication Nos. 2008/0299580; 2008/0159996; and 2010/0218264.

When endogenous sequences (endogenous or part of the transgene) are expressed with the transgene, the endogenous sequences (e.g., GAPDH, etc.) may be full-length sequences (wild-type or mutant) or partial sequences.

Preferably the endogenous sequences are functional. Non-limiting examples of the function of these fell length or partial sequences (e.g., GAPDH) include increasing the enzymatic activity of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier. Furthermore, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.

The invention will be described by the following non-limiting examples.

HDR occurs more efficiently with a chromatin donor template than with a naked DNA donor template in cells as disclosed herein. Although the examples employ the CRISPR-Cas9 RGN system, the chromatin HDR method could, however, be used in conjunction with any system (e.g., Cas9 or Cas9-related nucleases, other CRISPR-associated enzymes, TALENs, zinc finger nucleases, or other DNA sequence-specific cleaving reagents) for the generation of specific DSBs. Moreover, a chromatin HDR donor template can be used with other procedures for the enhancement of the efficiency of HDR. In this regard, the use of a chromatin HDR donor template would be one component of a multi- component system for HDR in cells.

Example 1

Methods Chromatin reconstitution

Native Drosophila core histones were purified as described (Fyodorov and Levenstein 2002; Khuong et al. 2017). The donor repair template plasmids were purified with the HiSpeed plasmid kit by following the manufacturer's recommendations (QIAGEN). The core histone:DNA ratio for each donor repair template was determined by carrying out a series of reactions with different core histone:DNA ratios and then assessing the quality of chromatin by the micrococcal nuclease digestion assay (Fyodorov and Levenstein 2002; Khuong et al. 2017). Chromatin was reconstituted with purified components by using the salt dialysis method (Stein 1989; Fei et al. 2015). In a typical chromatin reconstitution reaction, 50 pg plasmid DNA and 50 pg core histones were combined in TE buffer (10 mM Tris-HCl, pH 8, containing 1 tnM EDTA) containing 1 M NaCl in a total volume of 150 pL. The mixture was dialyzed at room temperature against the following buffers in the indicated order: 2 h in TE containing 0.8 M NaCl; 3 h in TE containing 0.6 M NaCl; 2.5 h in TE containing 50 ttiM NaCl. The quality' of the resulting chromatin assessed by using the micrococcal nuclease digestion assay, and the chromatin was stored at 4 °C until use.

Cell transfection

Cell transfection with chromatin or DNA donor templates was performed under the same conditions by following standard protocols. Transfection of HeLa cells was performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer's recommendations. Polyethylenimine (PEI; Biosciences) was used for transfection of MCF10A cells at a PEI:DNA mass ratio of 3:1. The transfections were performed as follows. 5xl0³ cells/well were plated in six well plates the day before transfection. For each CRISPR-Cas9 target locus, cells were co-transfected with equal amounts of the target-specific donor repair template (as free plasmid DNA or chromatin) and the Cas9 coding plasmid containing the target-specific single guide RNA sequence. For HeLa cells, 1.25 pg of DNA or chromatin was used; for MCF 10A cells, 1.5 pg of DNA or chromatin was used.

Results

Fig. 1A shows the region of the HDR donor construct (HDR donor) that was used for the targeted insertion of the T2A-GFP DNA sequence into tire human GAPDH gene. The GAPDH gene before ( GAPDH Locus) and after (GAPDH Edited Locus) the targeted insertion is shown. The GFP sequence is inserted close to the 3' untranslated region of Exon 9 (E9) of the GAPDH gene. Therefore, the targeted insertion of the GFP sequence does not result in an alteration of the GAPDH protein. Thus, after the targeted insertion of the GFP sequence, the wild-type GAPDH protein is produced along with a separate GFP protein.

Fig. IB shows the region of the HDR donor construct (HDR donor) that was used for the targeted insertion of the GFP DNA sequence into the human RAB11A gene. The RAB11A gene before (RAB11A Locus) and after (RAB11A Edited Locus) the targeted insertion is shown. The GFP sequence is inserted inframe into the coding region of Exon 1 (El) of the RAB11A gene. Thus, the targeted insertion of the GFP sequence results in the production of a GFP- RAB 11 A fusion protein.

Fig. 1C shows the region of the HDR donor construct (HDR donor) that was used for the targeted insertion of the GFP DNA sequence into the human ACTB gene. The ACTB gene before ( ACTB Locus) and after (ACTB Edited Locus) the targeted insertion is shown. The GFP sequence is inserted in-frame into the coding region of Exon 2 (E2) of the ACTB gene. Thus, the targeted insertion of the GFP sequence results in the production of a GFP-ACTB fusion protein.

The three constructs shown in Figures 1A, IB, and 1C were used in the experiments shown in Figures 2, 3, 4, 5, and 6.

The GFP (green fluorescent protein) and mEGFP (monomeric enhanced green fluorescent protein) DNA sequences were used became they produce the GFP protein, which can be detected by fluorescence-based flow cytometry and other fluorescence detection methods.

Notably, the successful incorporation of the GFP DNA sequences into the cells can be detected by the appearance of the GFP protein, which is detected by its fluorescence. That is, the edited cells that have incorporated the GFP DNA sequences could be identified by their green fluorescence.

Three different loci were tested to determine whether the chromatin HDR donor template can enhance HDR at different genomic regions. As seen later, it was found that chromatin enhances HDR at all three loci.

Example 2

Fig. 2 shows the overall design and strategy of the HDR experiments. Fig. 2A shows the two plasmid DNA constructs that were used in the HDR experiments, which are as follows, (i) The Cas9-T2A-mCherry & sgRNA plasmid contains sequences that encode the Cas9 and the mCherry proteins joined by the T2A self-cleaving peptide sequence. This configuration allows the translation of Cas9 and mCherry (a red fluorescent protein) as a single polypeptide that is subsequently autocleaved to generate the separate Cas9 and mCherry proteins. Thus, the expression of Cas9-T2A -mCherry fusion protein in the cell can be detected by the red fluorescence of mCherry'. The Cas9-T2A- mCherry & sgRNA plasmid also contains the DNA sequences that produce the sgRNA (single guide RNA). The sgRNA binds to the Cas9 protein, and the resulting complex mediates the sequence-specific cleavage of the target locus, which is complementary to the sequence in the sgRNA. The sgRNA provides the sequence-specificity of the DNA cleavage. A different sgRNA sequence is needed for each of the three target loci ( GAPDH , RABllA, aaAACTB) used in this work. Thus, three different Cas9-T2A-mCherry & sgRNA plasmids (e.g., one that is specific for GAPDH, one that is specific for RABllA, and one that is specific iorACTB) were used in this work. The Cas9-T2A-mCherry plasmid was transfected as naked DNA, not as chromatin. In this study, only the HDR donor construct was assembled into chromatin.

(ii). The HDR Donor Template contains the GFP sequences and flanking homology arms (5¹ HA and 3' HA) that will be used for the targeted insertion of the GFP DNA sequences into the target loci (GAPDH, RABllA, and ACTB ), which are shown in Fig. 1A, IB, and 1C. In this study, we test the relative efficiency of HDR when the HDR Donor Template is transfected into the cells as chromatin or naked DNA. There are three HDR Donor Templates used in this work. The first Donor Template is specific for GAPDH and contains the sequences shown in Fig. 1A. The second Donor Template is specific for RABllA and contains the sequences shown in Fig. IB. The third Donor Template is specific for ACTB and contains the sequences shown in Fig. 1C.

Fig. 2B shows the reconstitution of chromatin from naked DNA and purified core histone proteins. Chromatin was reconstituted by using the salt dialysis method, as described by Stein (1989). The hatched yellow circles in the chromatin denote nucleosomes, which are the repeating units of chromatin. Although chromatin was prepared by using the salt dialysis method, there are many different methods for making chromatin (see, for example: Lusser and

Kadonaga, 2004; Khuong et al., 2017). The invention described in this patent application is not restricted to any particular method for the preparation of chromatin.

Fig. 2C shows the three HDR Donor Templates (as shown and described in Fig. 2A) after reconstitution into chromatin. The chromatin was analyzed by the micrococcal nuclease digestion assay. The ladder of bands labeled "mono", "di", "tri", "tetra", and "penta" refer to the bands that are derived from

mononucleosomes, dinucleosomes, trinucleosomes, tetranucleosomes, and pentanucleosomes, respectively. The presence of distinct bands indicates that there was efficient assembly of nucleosomes onto each of the three HDR Donor Templates.

Fig. 2D shows the outline of the workflow in the CRISPR-Cas9-mediated editing experiments with chromatin or naked DNA donor templates. In these experiments, the chromatin HDR donor templates and the naked DNA HDR donor templates were tested in parallel to allow a direct comparison between the chromatin versus naked DNA HDR donor templates. The steps are as follows, (i). The Cas9-T2A-mCherry & sgRNA plasmid DNA (which contains the coding sequences for Cas9 and mCherry and a target-specific sgRNA sequence; see Fig. 2A) was co-transfected into human cells (either MCF 10A or HeLa cells) with the corresponding HDR Donor Template (see Fig. 2A) as naked DNA or chromatin (see Figs. 2B and 2C). (ii). At 24 hours post-transfection, cells that express the Cas9 and mCherry proteins (as detected by the red fluorescence of mCherry) were enriched by FACS (fluorescence activated cell sorting). The presence of the red fluorescence indicates the successful introduction of the

Cas9-T2A-mCherry & sgRNA plasmid into the cell and the likely co- transfection of the HDR Donor Template (as either naked DNA or chromatin) into the same cells. The red fluorescent cells were cultured (grown) for an additional 10 days to allow time for the removal/loss of flee HDR Donor Template constructs that were not incorporated into the genomic DNA of the cells. The free (i.e. , not incorporated into the genome) HDR Donor Templates contain GFP DNA sequences can also transiently produce low levels of GFP protein that would interfere with the next step, which is the detection of GFP- positive cells that exhibit green fluorescence, so the cells are cultured to allow for the removal/loss of any free HDR donor template, (iii). For each sample,

200,000 cells were subjected to flow cytometry analysis. GFP-positive (i.e., green fluorescent) cells in which the GFP sequences were stably incorporated into the genome by HDR were sorted by FACS. This step provided two important functions.

Function #1: The analysis by flow cytometry allowed the determination of the percentage of cells that were GFP-positive. This is important information that is shown in Figs. 3A, 3B, 3C, 5A, and 5B. Function #2: The FACS was able to sort individual GFP-positive cells into single wells in 96 well plates. This enabled the growth of individual clones that each derive from a single cell. These cells were cultured and their genomic DNA was analyzed by PCR, as shown in Figs. 4A, 4B, 4C, 5C, 5D, and 5E.

These experiments were performed under standard CRISPR-Cas9 genome editing conditions, such as those described by Ran et al. (2013), except that w chromatin HDR Donor Templates were used in addition to standard naked DNA HDR Donor Templates.

Example 3

Fig. 3 shows the results of the experiments described in Fig. 2D, step (iii), Function #1. These experiments were performed with human MCF10A cells, which are diploid. MCF10A cells are non-tumorigenic epithelial cells derived from human mammary glands.

Fig. 3A shows the flow cytometry analysis of the transfected cells, as described in Fig. 2D [step (iii), Function #1] For each sample, 200,000 cells were analyzed. The GFP-positive cells contain stably integrated GFP sequences. The percentage of GFP-positive cells reflects the efficiency of HDR. In each flow cytometry figure, the X-axis is FSC-A (forward scatter area, which reflects the cell size), and the Y-axis is the log of the intensity of the GFP fluorescence. The incorporation of GFP into the chromosomes results in GFP fluorescence and a higher position on the Y-axis than that of control cells that do not contain the GFP sequence (Untransfected). The cells in the upper inset box of each figure are GFP-positive, indicating that they have a high level of GFP fluorescence that is distinctly above the background signal. In each of the upper inset boxes, the percentage of GFP-positive cells is indicated.

Fig. 3A (upper panel) shows a control background experiment in which the cells were not transfected. These cells lack GFP-positive cells (0% GFP-positive cells).

Fig. 3A (middle panels) show transfection experiments that were performed as in Fig. 2D, step (iii), Function #1, in which there was a naked DNA donor template. The GAPDH, RAB11A, andACTB donor constructs were each tested separately.

Fig. 3A (bottom panels) show transfection experiments that were performed as in Fig. 2D, step (iii), Function #1, in which there was a chromatin donor template. The GAPDH, RAB11A, and ACTB donor constructs were each tested separately. At the GAPDH locus, there was a higher efficiency of HDR with a chromatin donor template (1.43% GFP-positive cells) than with a naked DNA donor template (0.22% GFP-positive cells) in MCF10A cells.

At the RAB11 A locus, there was a higher efficiency of HDR with a chromatin donor template (9.94% GFP-positive cells) than with a naked DNA donor template (3.55% GFP-positive cells) in MCF10A cells.

At the ACTB locus, there was a higher efficiency of HDR with a chromatin donor template (0.49% GFP-positive cells) than with a naked DNA donor template (0.18% GFP-positive cells) in MCF10A cells.

Fig. 3A shows three representative experiments that compare the efficiency of HDR with chromatin versus naked DNA donor templates in MCF 10A cells.

Fig. 3B shows the combined results of three independent experiments with each of the GAPDH, RAB11A, and ACTB loci in MCF10A cells. The average and standard deviation are indicated for each set of experiments.

At the GAPDH locus, there was a higher average efficiency of HDR with a chromatin donor template (1.3% GFP-positive cells) than with a naked DNA donor template (0.19% GFP-positive cells) in MCF10A cells in three independent experiments.

At the RAB11A locus, there was a higher average efficiency of HDR with a chromatin donor template (8.9% GFP-positive cells) than with a naked DNA donor template (3.3% GFP-positive cells) in MCF10A cells in three independent experiments.

At the ACTB locus, there was a higher average efficiency of HDR with a chromatin donor template (0.38% GFP-positive cells) than with a naked DNA donor template (0.16% GFP-positive cells) in MCF 10A cells in three independent experiments.

Fig. 3C shows the fold-enhancement of HDR with a chromatin donor template relative to a naked DNA donor template with each of the GAPDH, RAB11A, and ACTB loci in MCF10A cells. The graphs in Fig. 3C are based on data in Fig. 3B.

At the GAPDH locus, there was a 6.8-fold increase in the efficiency of HDR with a chromatin donor template relative to a naked DNA donor template in MCF10A cells. At the RABIJA locus, there was a 2.7-fbld increase in the efficiency of HDR with a chromatin donor template relative to a naked DNA donor template in MCF10A cells.

At the ACTB locus, there was a 2.3-fbld increase in the efficiency of HDR with a chromatin donor template relative to a naked DNA donor template in MCF10A cells.

Thus, at three different loci (GAPDH, RABIJA, and ACTB) in human MCF 10A cell s, there was a higher efficiency of HDR with chromatin donor templates than with naked DNA donor templates. Therefore, HDR with chromatin donor templates occurs with higher efficiency than HDR with naked DNA donor templates.

Example 4

MCF10A cells are diploid; hence, they have two copies of each chromosome. It was therefore important to determine whether the GFP DNA sequences were inserted into only one chromosome ("monoallelic" insertion) or into both chromosomes ("biallelic" insertion). For most applications, biallelic insertions in which both chromosomes have been modified by HDR are required.

To determine the frequency of monoallelic versus biallelic insertions, it was necessary to

examine individual cells. Therefore, clonal populations of genetically identical cells that are each derived from a single cell were generated. This goal was accomplished as outlined in Fig. 2D, step (iii), Function #2. In these

experiments, individual GFP-positive cells (i.e., cells containing GFP sequences stably incorporated into at least one chromosome) were sorted by FACS into single wells in 96 well plates. The individual cells were then grown into colonies (i.e., populations of genetically identical cells that were each derived from a single cells). Then, the genomic DNA of each clonal population was analyzed by PCR.

Fig. 4A shows the data of the PCR analysis of individual cell clones that were generated during CRISPR-Cas9-mediated HDR, as outlined in Fig. 2D, step (iii). Function #2. The genomic DNA was isolated from each cell clone, and then subjected to PCR analysis with primers that flank the 5' and 3' homology arm sequences in the location at which the GFP DNA is inserted (as shown in Fig. 1). The insertion of the GFP DNA sequence results in an increase in the size of the PCR product. Thus, the edited chromosomes give a larger PCR product than the wild-type (unedited) chromosomes. Because MCF10A cells are diploid (i.e., have two chromosomes), cell clones might have one edited chromosome and one wild-type chromosome (monoallelic) or two edited chromosomes (biallelic). Examples of monoallelic cell clones include GAPDH (DNA HDR donor; upper panel) clones 2, 3, 5, and 9. Examples of biallelic cell clones include GAPDH (DNA HDR donor; upper panel) clones 1, 8, and 10.

Fig. 4A shows representative data from 60 different cell clones: (i). 10 clones; insertion of GFP at the GAPDH locus; DNA HDR donor; (ii). 10 clones; insertion of GFP at the GAPDH locus; Chromatin HDR donor; (iii). 10 clones; insertion of GFP at the RAB11A locus; DNA HDR donor; (iv). 10 clones;

insertion of GFP at the RAB11A locus; Chromatin HDR donor; (v). 10 clones; insertion of GFP at the ACTB locus; DNA HDR donor; (vi). 10 clones; insertion of GFP at the ACTB locus; Chromatin HDR donor. In addition, as a control, the genomic DNA from wild-type cells was also included (left lane of each panel).

It can be seen that there are biallelic and monoallelic clones as well as other clones that result from imperfect modifications of the chromosomes. The imperfect clones (denoted by asterisks) appear to lack a property edited chromosome, as indicated by either the absence of an edited chromosome or the presence of a PCR product whose size is not consistent with that of an edited or wild-type chromosome. The imperfect clones are undesirable side products that were generated timing the HDR experiments.

Fig. 4A contains only a subset of the PCR data. Fig. 4B is a summary of all of the PCR analysis data. (i). 53 clones; insertion of GFP at the GAPDH locus; DNA HDR donor; (ii). 52 clones; insertion of GFP at the GAPDH locus;

Chromatin HDR donor; (iii). 89 clones; insertion of GFP at the RAB11A locus; DNA HDR donor; (iv). 97 clones; insertion of GFP at the RAB11A locus;

Chromatin HDR donor; (v). 72 clones; insertion of GFP at the ACTB locus;

DNA HDR donor; (vi). 71 clones; insertion of GFP at the ACTB locus;

Chromatin HDR donor

Fig. 4C shows the frequency (as a percentage) of occurrence of biallelic, monoallelic, and imperfect HDR clones at the GAPDH, RAB11A, and ACTB loci in MCF10A cells. Fig. 4C (left panel; GAPDH locus) shows that there were 52% biallelic clones obtained with a chromatin HDR donor template and 26% biallelic clones obtained with a naked DNA donor template.

Fig. 4C (middle panel; RAB11A locus) shows that there were 54% biallelic clones obtained with a chromatin HDR donor template and 49% biallelic clones obtained with a naked DNA donor template.

Fig. 4C (right panel; ACTB locus) shows that there were 4% biallelic clones obtained with a chromatin HDR donor template and 0% biallelic clones obtained with a naked DNA donor template.

At three different loci (GAPDH, RAB11A, and ACffl) in human MCF10A cells, there was a higher frequency of biallelic HDR insertions with chromatin donor templates than with naked DNA donor templates. Therefore, biallelic HDR insertions can be achieved with higher frequency with chromatin donor templates than with naked DNA donor templates.

In addition, to confirm the integrity of the biallelic clones (i.e., to confirm that these clones have the exact DNA sequence as predicted), three independent GAPDH biallelic clones were sequenced across the 5' and 3' junctions between the genomic GAPDH sequences and the inserted GFP sequences. For all three clones, the DNA sequence analysis showed that the GFP sequences were precisely inserted into the GAPDH gene as predicted.

Example 5

The data shown in Figs. 3 and 4 were obtained in human MCF 10A cells. The effects of a chromatin donor template on the efficiency of HDR was then tested in a different cell line - human HeLa cells. HeLa cells are human cervical adenocarcinoma cells that have been widely used in biomedical research. Unlike the diploid MCF10A cells, however, HeLa cells are aneuploid - that is, they contain abnormal numbers of chromosomes. For example, the GAPDH gene is located on human chromosome 12, and HeLa cells have four copies of chromosome 12.

To test whether a chromatin donor template enhances HDR in a different cell line, HDR experiments were performed with the CRISPR-Cas9 system, as outlined in Fig. 2D [step (iii), Function #1], except that the mCherry-positive cells were not enriched. Notably, HeLa cells have four copies of the GAPDH gene. Fig. 5A shows that there was a higher average efficiency of HDR with a chromatin donor template (4.3% GFP-positive cells) than with a naked DNA donor template (2.1% GFP-positive cells) with the GAPDH gene in HeLa cells in three independent experiments. These experiments were performed as in Fig. 3 with HeLa cells instead of MCF10A cells.

Fig. 5B shows a 2 fold-enhancement of HDR with a chromatin donor template relative to a naked DNA donor template at the GAPDH locus in HeLa cells. The graph in Fig. 5B is based on data in Fig. 5A.

Fig. 5C shows the data of the PCR analysis of individual cell clones that were generated dining CRISPR-Cas9-mediated HDR, as outlined in Fig. 2D, step (iii). Function #2. These experiments were performed as in Fig. 4A with HeLa cells instead of MCF10A cells.

Fig. 5C shows representative data from 20 different cell clones: (i). 11 clones; insertion of GFP at the GAPDH locus; DNA HDR donor; HeLa cells; (ii). 9 clones; insertion of GFP at the GAPDH locus; Chromatin HDR donor;

HeLa cells.

HeLa cells have four copies of the GAPDH gene. Therefore, the homozygous clones (with only properly edited chromosomes; such as in lanes 16 and 20) have four copies of the integrated GFP sequence, and the heterozygous clones (with both edited chromosomes and wild-type chromosomes; such as in lanes 1, 3, 4, 7, 8, 10, 11, 12, 13, 15, 18, and 19) have one to three copies of the integrated GFP sequence. There were also imperfect insertions (indicated by asterisks) in which the chromosomes were not properly edited.

As noted, Fig. 5C contains only a subset of the PCR data. Fig. 5D is a summary of all of the PCR analysis data. (i). 21 clones; insertion of GFP at the

GAPDH locus; DNA HDR donor; HeLa cells; (ii). 18 clones; insertion of GFP at the GAPDH locus; Chromatin HDR donor; HeLa cells

Fig. 5E show's the frequency (as a percentage) of occurrence of homozygous, heterozygous, and imperfect HDR clones at the GAPDH locus in HeLa cells. There were 28% homozygous clones obtained with a chromatin HDR donor template and 5% homozygous clones obtained with a naked DNA donor template at the GAPDH locus in HeLa cells.

The efficiency of HDR in HeLa cells is higher with chromatin donor templates than with naked DNA donor templates. These experiments show that the enhancement of HDR by the use of chromatin donor templates is observed in different cell lines. The generation of homozygous edited cells results from the insertion of the GFP DNA sequences into all four copies of the GAPDH gene. This is an impressive technical accomplishment.

Example 6

Fig. 6A shows that shows that the introduction of chromatin into MCF10A cells is less deleterious than the introduction of naked DNA into cells. At 24 h after transfection of the GAPDH HDR donor construct as either naked DNA or chromatin, the viability of chromatin-transfected cells (88.4%) is higher than the viability of naked DNA-transfected cells (77.3%).

Fig. 6B shows that shows that the introduction of chromatin into HeLa cells is less deleterious than the introduction of naked DNA into cells. At 3 days after transfection of the GAPDH HDR donor construct as either naked DNA or chromatin, the viability of chromatin-transfected cells (52.1%) is much higher than the viability of naked DNA-transfected cells (32.4%) .

Why might chromatin be less deleterious to cells than naked DNA? Because the natural form of DNA in the eukaryotic nucleus is chromatin and not naked DNA, it is reasonable that chromatin is less toxic to cells than naked DNA.

The introduction of chromatin into cells is less toxic than the introduction of naked DNA into cells. This is another advantage to the use of chromatin HDR donor templates relative to naked DNA HDR donor templates.

In summary, these experiments show that there is a higher efficiency of HDR as well as a higher viability of cells throughout the HDR process by the use of a natural chromatin donor template rather than a naked DNA donor template. These effects are likely due to the use of the natural chromatin form of the donor template rather than the unnatural naked DNA.

The enhancement of HDR by a chromatin template was tested with three different loci {GAPDH, RAB11A, andACTB ) in two different cell lines

(MCF10A, HeLa). The chromatin donor template enhanced the overall efficiency of HDR by 2- to 6.8-fold, as measured by the incorporation of GFP by flow cytometry (Figs. 3A, 3B, 3C, 5A, 5B). We never observed less than a 2- fold increase in HDR with a chromatin donor template reative to a naked DNA donor template. In addition, a chromatin donor template enhanced the biallelic insertion of GFP sequences into the specific target loci in MCF10A cells (Figs. 4A, 4B, 4C) and the tetraallelic insertion of GFP sequences into the GAPDH locus in HeLa cells (Figs. 5C, 5D, 5E)

These experiments further revealed that chromatin is less deleterious to cells than naked DNA in transfection experiments with HeLa cells and MCF 1 OA cells (Figs. 6A and 6B).

Therefore, the use of chromatin for HDR experiments results in three advantages: (i) higher efficiency of HDR with chromatin relative to naked DNA; (ii) higher frequency of biallelic HDR in diploid cells with chromatin donor templates relative to naked DNA donor templates; and (iii) higher cell viability after chromatin transfection than after naked DNA transfection.

Importantly, a chromatin HDR donor template can be used in conjunction with other methods for the enhancement of the efficiency of HDR In this regard, the use of a chromatin HDR donor template would be one component of a multi-component system for HDR in cells.

Although these experiments were performed in human cells, the native form of DNA in the nuclei of all eukaryotes is chromatin. In addition, the factors that are involved in HDR are present in all eukaryotes. Thus, the concept that HDR occurs more efficiently with chromatin than with naked DNA is applicable to all eukaryotes.

This method does not involve any special treatment (such as the addition of drags, or cold shock, or cell cycle synchronization) or genetic modification of the cells. Therefore, this method can be used on all types of cells.

In addition, HDR experiments often feil because of the cell death that is caused by the addition of naked DNA to cells. This is a problem, in particular, with cells that can be obtained only in small numbers. Therefore, the use of the disclosed method may be the difference between success and failure of HDR with cells, such as some primary cells, that can be obtained only in small quantities. References

Achen et al., Growth Factors. 20:99 (2002).

Alexiadis and Kadonga, Genes Dev..16:2767 (2002).

Bergers and Hanahan, Nat. Rev. Cancer. 8:592 (2008).

Beumer et al., Proc. Natl. Acad. Sci. USA, 105: 19821 (2008).

Booij et al., Cardiovasc. Res.. 111:217 (2016).

Bottsfoid-Miller et al., J. Clin. Oncol. 30:40264 (2012).

Bozas et al., Genetics. 182:641 (2009).

Canny et al., Nat. Biotechnol.. 36:95 (2018).

Carroll, Annu. Rev. Biochem.. 83:409 (2014).

Chang et al.. Proc. Natl. Acad. Sci. USA 84:4959 (1987).

Charpentier et al., Nat. Cotnmun.. 9: 1133 (2018).

Chu et al., Nat. Biotechnol.. 33:543 (2015).

Cong et al., Sciencexpress/10.1126/science.1231143 (2013). Fei et al., Genes Dev.. 29:2563(2015).

Fyodorov and Levenstein, Curr. Protoc. Mol, Biol. 21. unit 21.7 (2002) Folkman, Eur. J. Cancer Clin. Oncol.. 23:361 (1987).

Gao et al., J. Biol. Chem.. 283:7834 (2008).

Gao et al., J. Biol. Chem.. 285:28097 (2010).

Gao et al., Nat. Cell Biol.. 8: 1171 (2006).

Gilbert et al., N. Engl. J. Med.. 370:699 (2014).

Gonzalez and McGraw, Cell Cycle. 8:2502 (2009).

Guo et al., Am, J. Cancer Res.. 7:2234 (2017).

Guo et al., Sci. Ren.. 8:2080 (2018).

Gutschner et al., Cell Rep.. 14:1555 (2016).

Hanahan and Weinberg, Cell. 100:57 (2000).

Harrison et al., Genes Dev.. 28: 1859 (2014).

Hurwitz et al., N. Engl. J. Med.. 350:2335 (2004).

Jayson et al., Lancet. 388:518 (2016).

Jinek et al., Science 337:816 (2012). Kato-lhui et al., Nucleic Acids Res.. 46:4677 (2018).

Kerbel, N. Enel. J. Med.. 358:2039 (2008).

Khuong et al., J. Biol. Chem.. 292:19478 (2017).

King et al., PLoS One. 6:el8713 (2011).

Kitchinc et al.. Biochim. Bionhvs. Acta.. 1625: 116 (2003V Li et al. Nucleic Acids Res.. 46:10195 (2018).

Lin et al., Oncogene. M:384 (2015).

Lin et al., Elife. 3:e04766 (2014).

Liu et al., J. Mol. Cell Caidiol.. 72:28 (2014).

Lusser and Kadonaga, Nat. Methods L 19 (2004).

Ma et al, EXP. Cell Res.. 362: 142 (2018).

Mo et al., Am. J. Transl. Res.. 8:4951 (2016).

Nakagawa et al., Oncotarget. 6:3825 (2015).

Nehls et al., Science 272:886 (1996). Ohgaki, Methods Mol. Biol.. 472:323 (2009).

Ostrom et al.. Cancer Treat. Res.. 163: 1 (2015).

Paduch, Cell Oncol. (Dordri. 39:397 (2016).

Paranjape et al., Annu. Rev Biochem.. 63:265 (1994).

Perez Pinero et al., Br. J. Pharmacol .. 166:721 (2012).

Pinder et al., Nucleic Acids Res.. 43:9379 (2015).

Prasad et al., Mol Cell Biochem.. 336:25 (2010).

Quadras et al. Genome Biol..18:92 (2017).

Ran et al., Nat. Protoc.. 8:2281 (2013).

Robert et al., Genome Med.. 7:93 (2015).

Roth et al., Nature. 559:405 (2018).

Sanchez-Rivera and Jacks, Nat. Rev. Cancer. 15:387 (2015). Sastri et al., Proc. Natl. Acad. Sci. U.S.A.. 102:349 (2005). Sastri et al., Proc. Nad. Acad. Sci. U S A.. 110:E387 (2013). Sharma et al- Nano. Lett.. 17:1356 (2017).

Stein, Methods Enzvmol., 170:585 (1989). Takayama et al., Nucleic Acids Res.. 45:5198 (2017).

Tebbutt et al., J. Clin. Oncol.. 28:3191 (2010).

Viatour et al., Trends Biochem. Sci.. 30:43 (2005).

Yang et al., Sci. Ren.. 6:21264 (2016).

Zhang et al.. Genet. Mol. Res.. 13: 10648 (2014).

Zhang et al., Genome Med.. 5:33 (2013).

Zhang et al., Genome Biol., 18:35 (2017).

Zhong et al., cell 89:413 (1997).

Zhong et al., Mol. Cell 1:661 (1998).

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method to enhance the efficiency of homology directed repair in cells, comprising: contacting cells with an effective amount of isolated nucleic acid complexed as chromatin to be employed in homology directed repair and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease, under conditions allowing for enhanced homology directed repair in one or more chromosomes of the cells relative to cells contacted with isolated nucleic acid that is not complexed as chromatin to be employed in the homology directed repair and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease.

2. The method of claim 1 wherein the nucleic acid complexed as chromatin encodes a protein.

3. The method of claim 1 or 2 wherein the nuclease comprises a Cas

protein, a ZFN or a TALEN.

4. The method of any one of claims 1 to 3 wherein the cells are from a mammal.

5. The method of claim 4 wherein the mammal is a human.

6. The method of claim 4 wherein the mammal is a bovine, caprine, ovine, swine, canine, feline, or non-human primate.

7. The method of any one of claims 1 to 6 wherein the repair results in an insertion.

8. The method of any one of claims 1 to 7 wherein the repair results in a deletion.

9. The method of any one of claims 1 to 8 wherein the repair results in replacement of a sequence in the cells.

10. The method of any one of claims 1 to 9 wherein the cells are plant cells or animal cells.

11. The method of claim 3 wherein the nuclease is encoded on a plasmid that is contacted with the cell.

12. The method of claim 11 wherein the plasmid further comprises guide RNA.

13. The method of any one of claims 1 to 10 wherein the cells are contacted with a composition comprising nucleic acid complexed as chromatin to be employed in the homology directed repair and the nuclease.

14. The method of any one of claims 1 to 10 wherein the cells are contacted with a composition comprising nucleic acid complexed as chromatin to be employed in the homology directed repair and isolated nucleic acid encoding the nuclease.

15. The method of any one of claims 1 to 14 wherein the length of the

isolated nucleic acid to be employed in the homology directed repair is at least 150 bp.

16. The method of any one of claims 1 to 15 wherein the length of the

isolated nucleic acid to be employed in the homology directed repair is less than about 50 kbp.

17. The method of any one of claims 1 to 16 wherein the enhancement is at least 2-fold.

18. The method of any one of claims 1 to 17 wherein about 150 to 200 bp of isolated nucleic acid is complexed with 2 molecules of each of the core histones H2A, H2B, H3, and H4.

19. The method of claim 18 wherein the histones are mammalian histones.

20. The method of claim 18 wherein the histones are human histones.

21. The method of any one of claims 1 to 20 wherein the homology directed repair is biallelic.

22. The method of any one of claims 1 to 21 wherein the isolated nucleic add comprises mammalian DNA.

23. The method of claim 22 wherein the DNA comprises human DNA.

24. An in vivo method to enhance the efficiency of homology directed repair in a plant or animal, comprising: contacting cells of the plant or administering to the animal an effective amount of a composition comprising isolated nucleic acid complexed as chromatin to be employed in homology directed repair and a nuclease useful in gene editing or isolated nucleic acid encoding the nuclease.

25. The method of claim 24 wherein the administration is local.

26. The method of claim 24 wherein the administration is systemic.

27. The method of any one of claims 24 to 26 wherein the isolated nucleic acid has at least one nucleotide substitution, insertion or deletion relative to nucleic acid in the cells of the plant or animal.

28. The method of any one of claims 24 to 27 wherein the nucleic acid

encodes a protein.

29. The method of any one of claims 24 to 28 wherein the nuclease

comprises a Cas protein, a ZFN or a TALEN.

30. The method of any one of claims 24 to 29 wherein the animal is a

mammal.

31. The method of claim 30 wherein the mammal is a human.

32. The method of claim 30 wherein the mammal is a bovine, caprine, ovine, swine, canine, feline, or non-human primate.

33. The method of any one of claims 24 to 32 wherein the repair results in an insertion.

34. The method of any one of claims 24 to 33 wherein the repair results in a deletion.

35. The method of any one of claims 24 to 34 wherein the repair results in replacement of a sequence in the cells.

36. The method of any one of claims 24 to 35 wherein the nuclease is

encoded on a plasmid.

37. The method of claim 36 wherein the plasmid further comprises guide RNA.

38. The method of any one of claims 24 to 37 wherein the length of the isolated nucleic acid to be employed in the homology directed repair is at least 150 bp.

39. The method of any one of claims 24 to 38 wherein the length of the isolated nucleic acid to be employed in the homology directed repair is less than about 50 kbp.

40. The method of any one of claims 24 to 39 wherein the enhancement is at least 2-fold.

41. The method of any one of claims 24 to 40 wherein about 150 to 200 bp of isolated nucleic acid is complexed with 2 molecules of each of the core histones H2A, H2B, H3, and H4.

42. The method of claim 41 wherein the histones are mammalian histones.

43. The method of claim 41 wherein the histones are human histones.

44. The method of any one of claims 24 to 43 wherein the homology directed repair is biallelic.

45. The method of any one of claims 24 to 44 wherein the isolated nucleic acid comprises mammalian DNA.