Patent Description:
Targeted genome editing is an emerging and important technology with broad research and therapeutic applications. Customizable nucleases can be used to make targeted double-stranded breaks (DSB) in living cells, the repair of which can be exploited to induce desired sequence changes.

Two competing pathways effect repairs in most cells, including mammalian cells. Repair of a nuclease-induced DSB by non-homologous end-joining (NHEJ) leads to the introduction of insertion/deletion mutations (indels) with high frequencies. By contrast, DSB repair by homology directed repair (HDR) with a user-supplied "donor template" DNA can lead to the introduction of specific alterations (e.g., point mutations and insertions) or the correction of mutant sequences back to wild-type.

The present invention is recited in the claims. The invention is based on the development of methods for improving the absolute rate of homology-directed repair (HDR) and/or the relative rate of HDR compared with non-homologous end joining (NHEJ).

Thus, in one aspect which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include contacting the cell with or expressing in the cell (i) a double-stranded region of a donor nucleic acid molecule comprising the specific sequence to be inserted into the target nucleic acid and (ii) a DNA binding domain (DBD), e.g., an engineered DNA binding domain, that binds to or near (e.g., within <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> nucleotides of) the target site, wherein the DBD also binds to a double-stranded region of a donor nucleic acid molecule comprising the specific sequence to be inserted into the target nucleic acid; and inducing a double stranded break (DSB) at the target site, under conditions sufficient for the donor nucleic acid molecule to be inserted into the site of the DSB and the DSB to be repaired, thereby introducing the specific sequence into the target site.

In some embodiments of the disclosure, the DBD is a zinc finger domain, a transcription-activator-like effector (TALE) domain, or a "dead" Cas9 variant lacking nucleases activity ("dCas9"), that binds directly to a double-stranded DNA portion of the donor molecule that is near (e.g., within <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> nucleotides of) the target site.

In some embodiments of the disclosure, the nuclease is ZFN, TALEN, or Cas9 protein.

In some embodiments of the disclosure, the methods include expressing a fusion protein comprising a DBD linked, e.g., via an optional intervening linker of from <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> amino acids, to the nuclease used to make the DSB.

In some embodiments, the DBD is linked to a second DBD that binds adjacent to or near, e.g., within <NUM>, <NUM>, <NUM>, or <NUM> nts of, the target DSB site.

In some embodiments of the disclosure, the donor molecule is wholly double-stranded donor template or partially double-stranded and partially single-stranded DNA.

In some embodiments of the disclosure, the methods include expressing in the cell: a first fusion protein comprising a dimerization domain and the DBD that binds to a donor nucleic acid molecule (rather than covalent linkages), and a second fusion protein comprising a corresponding dimerization domain and a nuclease that induces a DSB at the target site or a second DBD that binds a DNA sequence adjacent to the target DSB.

In some embodiments of the disclosure, the DBD that binds the donor molecule is fused or bound to Csy4, the nuclease is Cas9, and the guideRNA is fused to a Csy4 recognition sequence.

In some embodiments of the disclosure, the nuclease is fused to a catalytically inactive Csy4 (dCsy4), and the donor molecule is a RNA-DNA hybrid comprising a Csy4 recognition site (RNA) and a double-stranded donor (DNA).

In another aspect, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include expressing in the cell a nuclease that induces DSBs only in specific phases of the cell cycle, comprising a fusion protein comprising a cell-cycle regulated protein domain linked to an engineered nucleases. The claimed composition comprises a nuclease that induces DSBs only in specific phases of the cell cycle, the nuclease comprising a fusion protein comprising a cell-cycle regulated protein domain linked to an engineered nucleases.

In embodiments of the claimed invention, the cell-cycle regulated protein domain is from a G2 or S-phase specific proteins, CtIP, Cdk2, Cyclin A1, Cyclin A2, Cyclin B1, or Gemini, e.g., amino acids <NUM>-<NUM> of human Gemini.

In some embodiments, the engineered nuclease is selected from the group consisting of a ZFN, a TALEN, a CRISPR/Cas9, and a CRISPR RNA-guided FokI nucleases (RFNs).

In some embodiments, the fusion protein is selected from the group consisting of hGem-ZFN, ZFN-hGem, mAG-hGem-ZFN, ZFN-mAG-hGem, hGem-TALEN, TALEN-hGem, mAG-hGem-TALEN, TALEN-mAG-hGem; hGem-Cas9, Cas9-hGem, mAG-hGem-Cas9, Cas9-mAG-hGem, hGem-Csy4, hGem-mAG-Csy4, Csy4-hGem, or Csy4-mAG-hGem, hGem-FokI-dCas9, hGem-mAG-FokI-dCas9, FokI-dCas9-hGem, FokI-dCas9-hGem-mAG, hGem- dCas9-FokI, hGem-mAG-dCas9-Fokl, dCas9-FokI-hGem, or dCas9-FokI-hGem-mAG.

In some embodiments, the constructs comprise one or more nuclear localization signals and nuclear export signals, or nuclear-cytoplasmic shuttle sequences to control the trafficking of nuclease proteins into the cytoplasm.

In another aspect which do not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA, the method comprising globally expressing one or more components of the HDR pathway throughout the cell cycle.

In some embodiments of the disclosure, the methods include contacting the cell with or expressing in the cell an engineered fusion protein comprising a transcriptional activation domains (e.g., VP64, VP16, NF-KB p65) and a sequence-specific DNA binding domains (e.g., engineered zinc fingers, TALEs, or dCas9 complexed with specific guide RNAs), to thereby upregulate a components of the HDR pathway.

In some embodiments of the disclosure, the factors to upregulate include one or more of Rad50, Rad51, Rad52, Rad54, BRCA1, or BRCA2.

In another aspect, which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include expressing in the cell an engineered nuclease (e.g., ZFN, TALEN, Cas9 nuclease, Cas9 nickase, or CRISPR RNA-guided FokI nuclease) to generate a DSB at the target site, and recruiting HDR factors to or blocking NHEJ factors from the same genomic site.

In some embodiments of the disclosure, the methods include expressing in the cell one or more of: a fusion protein comprising an HDR factor linked to a DBD that binds to a sequence near the target site, a fusion protein comprising an HDR factor linked to the engineered nuclease, a first fusion protein comprising an HDR factor linked to a dimerization domain and a second fusion protein comprising an engineered nuclease linked to a corresponding dimerization domain, a fusion protein comprising an HDR factor linked to an RNA-binding protein (e.g., MS2 or Csy4) that interacts with a specific RNA sequence appended to the end of a guide RNA sequence, and/or expression from a plasmid of any pro-HDR or anti-NHEJ factor.

In some embodiments of the disclosure, the HDR factor is selected from the group consisting of nucleases or helicases to process free DNA ends, and protein binding domains to act as nucleation sites for supplementary HDR factors.

In some embodiments of the disclosure, the HDR-related protein is selected from the group consisting of Nucleases and/or helicases that promote DNA strand resection, e.g., MRE11, EXO1, DNA2, CtIP, TREX2, and Apollo; Binding factors/nucleation proteins that recruit specific factors or catalyze strand invasion, e.g., BRCA1, BRCA2, PALB2, RAD50 orNBS1, RAD51, RAD52, RAD54, SRCAP, FANCI, FANCD2, BRIP1, SLX4, FANCA, FANCE, and FANCL (including truncated, mutated, modified, or optimized versions of these factors).

In another aspect, which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include locally blocking or binding NHEJ-associated factors, including transcriptional repression of pro-NHEJ factors.

In some embodiments, which does not form part of the claimed invention, the methods include expressing in the cell a fusion protein comprising a DBD that binds to or near the target site fused to a version of DNA-PK that interact and bind Ku70 but is impaired for recruitment of end-processing factors such as Artemis, polynucleotide kinase/phosphatase (PNKP), AP endonuclease <NUM> (APE1) and tyrosyl-DNA posphodiesterase (TDP1), or fused to a defective version of Rif1.

In another aspect, which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include inducing a double stranded break (DSB) at the target site, wherein the DSB has <NUM>' overhangs.

In some embodiments of the disclosure, the methods include expressing in the cell: pairs of engineered nickases (e.g., ZFNickases or Cas9 nickases) positioned to form a DSB with <NUM>' overhangs; one or more ZFN, TALEN, or CRISPR RNA-guided nucleases comprising dimerization-dependent nuclease domains that make DSBs with <NUM>' overhangs, e.g., a nuclease domain from Kpn I; or a fusion protein comprising a FokI cleavage domain fused to a Cas9 nickase (e.g., H840- or N863A-Cas9 nickase), and two guide RNAs spaced to generate a DSB with <NUM>' overhangs.

In another aspect, which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include expressing in the cell a pair of fusion proteins, each comprising an engineered DNA binding domain linked to Spo11 (in either N-, C-, or internal fusions), wherein each of the DBD-Spo11 monomer is targeted with appropriate spacing to create targeted DSBs with <NUM>' overhangs.

In another aspect, which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA. The methods include expressing in a cell a fusion protein comprising Cas9 and a chromatin modifier, e.g., SETD2, SRCAP, and SMARCAD1.

In another aspect, which does not form part of the claimed invention, the disclosure provides methods for introducing a specific sequence into a target site on a double-stranded nucleic acid in a cell, e.g., genomic DNA, by use of an in vitro produced protein-capped donor template.

In some embodiments, which do not form part of the claimed invention, the methods include expressing a Cas9-based nuclease or nickase, further comprising expressing in the cell one or more guide RNAs that bind to or near the target site.

In some embodiments, the absolute rate of homology-directed repair (HDR) and/or the relative rate of HDR as compared with the rate of non-homologous end joining (NHEJ) is improved.

Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions, will control.

A major unresolved challenge for genome editing is the inability to control whether a DSB is repaired by HDR with the donor template or by mutagenic NHEJ. HDR-mediated alterations can potentially be used to achieve the precise genome editing events that will be required for therapeutic applications, but the efficiencies with which these alterations are generally less efficient than NHEJ-mediated indels. Because alteration by HDR and NHEJ are competitive processes, indels can be introduced before desired precise changes. In addition, in some cases, secondary NHEJ-mediated indels can be introduced into alleles that have been corrected by HDR.

A method that would enable HDR to become more efficient than NHEJ or a method that suppressed NHEJ-mediated repair would broaden the scope of applications for nuclease-induced genome editing. Here we describe a number of strategies for increasing the absolute and relative rates of HDR by customizable nucleases. Note that although we describe these strategies using the clustered regularly interspaced short palindromic repeat-CRISPR-associated (CRISPR-Cas9) system to induce DSBs, many of these strategies are generalizable for use with any customizable nuclease platform (e.g.-meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs)). Any strategies not falling within the scope of the claims are provided for illustration purposes only.

A first method to enhance HDR by targeted DNA cleavage is to use an engineered DNA binding domain to physically localize the HDR donor molecule to the site of the targeted DSB. The rationale for this approach is that increasing the local concentration of donor molecule around a DSB will drive the subsequent DNA repair reaction toward targeted HDR-mediated repair (because a homologous donor molecule is required for the reaction to proceed), where the desired reaction is:
<CHM>
and the competing NHEJ-mediated repair reaction is given by:
<CHM>
such that the velocity of reaction (<NUM>) will increase as a result of the increased concentration of donor, while reaction (<NUM>) will not be directly affected by this perturbation; however, a bias toward reaction (<NUM>) could cause reaction (<NUM>) to proceed less efficiently.

This can be achieved using a DNA binding domain (DBD), e.g., an engineered DNA binding domain with programmable specificity, such as a zinc finger domain, a transcription-activator-like effector (TALE) domain, a "dead" Cas9 variant lacking nucleases activity ("dCas9"), or other DNA-binding platforms, e.g., as described herein - that binds directly to a double-stranded DNA portion of the donor molecule and localizes it to the site of the DSB. This DBD directs DSB localization in the target DNA, e.g., in the genome, through direct fusion to a nuclease used to make the DSB (such as a ZFN, TALEN, or Cas9 protein; see, for example, <FIG>) or through fusion to a second engineered DNA binding domain (of the same or a different type) that has been engineered to bind to a sequence near the target DSB site in the target DNA, e.g., in the genome. In either case, the first DBD can bind to either a wholly double-stranded donor template, to a double-stranded portion of an otherwise single-stranded DNA donor molecule, or to a double-stranded portion of a DNA molecule whose single-strand portion hybridizes to a single-stranded portion of a donor molecule.

Other variations on this approach include the use of dimerization domains (rather than covalent linkages) to join together the DBD that binds to or recruits the donor molecule with either the DSB-inducing nuclease(s) or a second DBD that binds DNA sequence adjacent to the target DSB (<FIG>). These dimerization domains might constitutively interact with one another (e.g., leucine zipper motifs (<NPL>), Fc domains) or might interact only in the presence of an effector such as a small molecule or stimulation by light. A number of dimerization domains are known in the art, e.g., cysteines that are capable of forming an intermolecular disulfide bond with a cysteine on the partner fusion protein, a coiled-coil domain, an acid patch, a zinc finger domain, a calcium hand domain, a CHI region, a CL region, a leucine zipper domain, an SH2 (src homology <NUM>) domain, an SH3 (src Homology <NUM>) domain, a PTB (phosphotyrosine binding) domain, a WW domain, a PDZ domain, a <NUM>-<NUM>-<NUM> domain, a WD40 domain, an EH domain, a Lim domain, an isoleucine zipper domain, and a dimerization domain of a receptor dimer pair (see, e.g., <CIT>; <CIT>; <CIT>; and <CIT>).

Suitable dimerization domains can be selected from any protein that is known to exist as a multimer or dimer, or any protein known to possess such multimerization or dimerization activity. Examples of suitable domains include the dimerization element of Gal4, leucine zipper domains, STAT protein N-terminal domains, FK506 binding proteins, and randomized peptides selected for Zf dimerization activity (see, e.g., <NPL>;<NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>). Furthermore, some zinc finger proteins themselves have dimerization activity. For example, the zinc fingers from the transcription factor Ikaros have dimerization activity (<NPL>). Thus, if the engineered Zf proteins themselves have dimerization function there will be no need to fuse an additional dimerization domain to these proteins. In some embodiments of the disclosure the endonuclease domain itself possesses dimerization activity. For example, the nuclease domain of Fok I, which has intrinsic dimerization activity, can be used (<NPL>). In some embodiments of the disclosure, "conditional" dimerization technology can be used. For example, this can be accomplished using FK506 and FKBP interactions. FK506 binding domains are attached to the proteins to be dimerized. These proteins will remain separate in the absence of a dimerizer. Upon addition of a dimerizer, such as the synthetic ligand FK1012, the two proteins will fuse.

Alternatively, the DBD that binds the donor molecule might be recruited to the DSB through fusion or non-covalent interaction with a factor that directly binds an RNA sequence or structure (e.g., an "RNA aptamer") appended to the Cas9 guide RNA (gRNA); for example, Csy4 can bind to a Csy4 recognition sequence that is fused to the gRNA.

Another variation on this approach would be to fuse a nuclease (such as a ZFN, TALEN, Cas9, or FokI-dCas9) to a RNA-binding domain such as catalytically inactive Csy4 (dCsy4, e.g., H29A Csy4 as described in <NPL>; <NPL>) and provide a RNA-DNA hybrid donor molecule consisting of Csy4 recognition site (RNA) and a standard donor (DNA). dCsy4 will bind to a Csy4 recognition site on the single stranded RNA-DNA hybrid donor and tether it in close local proximity to the targeted DSB. Alternatively, an RNA-binding domain such as MS2 could be used in place of Csy4. Notably, Cas9 has been previously reported to remain bound after cleavage, which makes it ideal for this application.

We note that, in some cases, non-programmable natural DNA domains might also be used in lieu of engineered DBDs to achieve similar ends to those described above.

NHEJ operates during all phases of the cell cycle, while HDR is restricted to the S and G2 phases of the cell cycle.

HDR machinery is regulated during the cell cycle and is present during S and G2 phases. DSBs created during M or G1 phases are preferentially repaired by NHEJ, while those made during S and G2 have the opportunity to be repaired by HDR. The expression of many endogenous cellular proteins are regulated in a cell-cycle specific manner by ubiquitination or phosphorylation-dependent degradation mechanisms. For example, the Geminin protein is degraded during the G1 phase of the cell cycle, but accumulates during S, G2, and M phases. Fusions of a fluorescent protein monomeric Azami Green (mAG) to portions of human Geminin (hGem) have been demonstrated to restrict fluorescence activity to S, G2, and M phases of the cell cycle; see, e.g., <NPL>; <NPL>);<NPL>); and <CIT>.

Here we describe methods to enhance HDR over NHEJ by the use of nucleases that induce DSBs only in specific phases of the cell cycle, that use fusions of cell-cycle regulated protein domains (e.g., hGEM, or amino acids <NUM>-<NUM> of hGEM) to engineered nucleases, such as ZFNs, TALENs, CRISPR/Cas9, and FokI-dCas9 or the Csy4 ribonuclease.

For ZFNs, in some embodiments exemplary fusion proteins would include hGem-ZFN, ZFN-hGem, mAG-hGem-ZFN, or ZFN-mAG-hGem.

For TALENs, in some embodiments exemplary fusion proteins would include hGem-TALEN, TALEN-hGem, mAG-hGem-TALEN, or TALEN-mAG-hGem.

For the wildtype CRISPR/Cas9 there are two components that can be regulated: Cas9 protein and the gRNA. First, exemplary Cas9 fusion proteins include hGem-Cas9, Cas9-hGem, mAG-hGem-Cas9, or Cas9-mAG-hGem. Second, activity of the gRNAs can be regulated by flanking with Csy4-recognition sites and placing it under control of a RNA Pol II promoter. In this context (see <CIT>), Cas9/gRNA activity depends on co-expression of and processing by the Csy4 ribonuclease. Placing Csy4 under cell cycle control (hGem-Csy4, hGem-mAG-Csy4, Csy4-hGem, or Csy4-mAG-hGem) is a potential strategy for regulating the activity of gRNAs by regulating the expression of Csy4.

For CRISPR RNA-guided FokI nucleases (RFNs), embodiments include hGem-FokI-dCas9, hGem-mAG-FokI-dCas9, FokI-dCas9-hGem, FokI-dCas9-hGem-mAG, hGem- dCas9-FokI, hGem-mAG-dCas9-FokI, dCas9-FokI-hGem, or dCas9-FokI-hGem-mAG.

Each of these constructs could additionally have combinations of nuclear localization signals and nuclear export signals, or nuclear-cytoplasmic shuttle sequences to control the trafficking of nuclease proteins into the cytoplasm, a critical step for ubiquitination and subsequent protein degradation. A PEST protein degradation tag, which is a peptide sequence that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (see, e.g., <NPL>)) could be added to generally reduce the half-life of the protein.

Instead of hGem, cell-cycle regulatory domains from other G2 or S-phase specific proteins could also be used, including: CtIP, Cdk2, Cyclin A1, Cyclin A2, and Cyclin B1. Preferably, human sequences would be used.

Additionally, cell-cycle-specific regulation could be achieved by expressing a nuclease such as Cas9, TALENs, or engineered zinc finger nucleases under the control of cell-cycle-specific transcription regulatory elements. Promoters or regulatory elements of genes controlled by the transcription factor E2F, such as Cyclin-A, Cyclin-E, and CDC2, could be used to express the nuclease during S phase only. The SV40 promoter has also been demonstrated to express primarily during S phase.

Individually, or in combination, these methods may restrict expression of the desired nuclease to the S and G2 phases of the cell cycle, thereby increasing the probability that the induced DSB is repaired by the HDR pathway.

Protein factors involved in the HDR pathway are regulated in a cell cycle-dependent manner. This restricts DSB repair by the HDR pathway to the S and G2 phases of the cell cycle, making precise alterations introduced by HDR inefficient relative to the indel-inducing NHEJ pathway. A strategy for more efficiently using HDR to make precise alterations to the genome is to globally express the critical components of the HDR pathway throughout the cell cycle. Specific methods for accomplishing this include using engineered fusions of transcriptional activation domains (e.g., VP64, VP16, NF-KB p65) to sequence-specific DNA binding domains (engineered zinc fingers, TALEs, or dCas9 complexed with specific guide RNAs) to upregulate critical components of the HDR pathway. Transcription activators that can be used in the TALE activators are known in the art, e.g., one or more, preferably four, VP16 peptides (e.g., the VP64 transcriptional activator sequence DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (SEQ ID NO:<NUM>), or an NF-KB p65 transactivation domain. See, e.g.,<NPL>; <NPL>; <NPL>; and <CIT>.

Factors that can be up-regulated include but are not limited to: Rad50, Rad51, Rad52, Rad54, BRCA1, BRCA2, and Apollo. Each of these factors can be upregulated alone or in any combination of the factors listed. For example, titrating BRCA1 expression may enhance HDR as it has been shown to inhibit the pro-NHEJ/anti-HDR role of 53BP1, resulting in greater activity of pro-HDR factors like CtIP at DSB sites.

Plasmids with the cDNA or mRNA encoding these factors could be transfected to transiently and globally upregulate these factors in cells.

The specific components of the two predominant DNA-repair pathways in cells (NHEJ or HDR) have been well studied, allowing individual components of either mechanism to be either recruited or blocked to influence the nature of the repair outcome. A customized nuclease (e.g., ZFN, TALEN, Cas9 nucleases, Cas9 nickases, CRISPR RNA-guided FokI nucleases) can be used to generate targeted DSBs while simultaneously recruiting HDR factors to or blocking NHEJ factors from the same genomic site.

DNA-repair via HDR involves multiple classes of proteins that include nucleases or helicases to process free DNA ends, and protein binding domains to act as nucleation sites for supplementary HDR factors. Examples of each class of HDR-related protein (and post-translationally modified derivatives of these factors) include but are not limited to the following:.

To enhance HDR, full length or truncated versions of any of the HDR-associated factors described here can be fused to either customized nucleases (<FIG>) or to a DBD that binds, e.g., is designed to bind, adjacent to the nuclease-induced DSB. As an alternative to covalently fusing these factors, they might also be directed to interact with a customized nuclease or an DBD via dimerization domains (<FIG>). In some instances, these dimerization domains might constitutively interact or interact in a temporally-restricted inducible fashion using either small molecule- or light-activated dimerization domains. Additionally, multiple distinct HDR factors could be recruited to the nuclease or DBD simultaneously (for example, Mre11, Rad50, and Nbs1 along with CtIP could all be localized to reconstitute the active MRN-CtIP complex). Alternatively, for Cas9 nucleases, Cas9 nickases, and/or CRISPR RNA-guided FokI nucleases, these HDR-associated factors might be fused to an RNA-binding protein (such as MS2 or Csy4) that can interact with a specific RNA sequence appended to the end of a guide RNA sequence (<FIG>). We note that various factors or dimerization domains might be fused to the customized nuclease AND a DBD in various combinations to induce HDR. Alternatively, RNA aptamers that bind specific HDR-associated factors might be fused to the <NUM>' end of the gRNA as single aptamers or in a combinatorial fashion to recruit these factors to a DSB (<FIG>). In the case of CRISPR RNA-guided FokI nucleases, the aptamers fused to one gRNA might differ from those fused to its paired gRNA, allowing for greater combinatorial recruitment of factors.

Additionally, any of the pro-HDR or anti-NHEJ factors listed herein can be overexpressed in the cell from a plasmid (as individual factors or combinations of factors) without covalent tethering to a nuclease, DBD, dimerization domain, or RNA-binding protein.

Error-prone NHEJ is the dominant DSB repair pathway in mammalian cells, is available during all phases of the cell cycle, and can repair DSBs with faster kinetics than the HDR pathway. One factor limiting the efficiency of HDR may be the rapid binding of Ku70, an NHEJ-factor, to DSBs, and the subsequent recruitment of DNA-dependent protein kinase (DNA-PK), 53BP1 (also called Tumor Protein P53 Binding Protein <NUM> or TP53BP1), and other critical components of the NHEJ machinery.

One approach to increase the efficiency of HDR includes recruitment of defective NHEJ machinery components, such as a version of DNA-PK that interact and bind Ku70 but is impaired for recruitment of one or more end-processing factors such as Artemis, polynucleotide kinase/phosphatase (PNKP), AP endonuclease <NUM> (APE1) and tyrosyl-DNA phosphodiesterase (TDP1).

53BP1 is also known to be a major regulator of DNA repair pathway choice. Recent studies have identified RAP1-interacting factor (Rif1) as an ATM phosphorylation-dependent interactor of 53BP1 that is the main factor used by 53BP1 to block <NUM>' end resection. One approach to impairing 53BP1 function would be to locally supply a defective version of Rif1. Additionally, it would be possible to down-regulate the expression of endogenous pro-NHEJ factors (including, but not limited to 53BP1) by targeting transcriptional repressors composed of KRAB or SID domains fused to dCas9 or other DBDs.

These strategies locally interfere with the canonical NHEJ pathway, thereby providing greater opportunity for <NUM>' end resection and homology directed repair to occur.

FokI-based nucleases such as ZFNs, TALENs, and recently described CRISPR RNA-guided FokI-nucleases (RFNs or fCas9) induce double-stranded breaks with a <NUM>-nucleotide <NUM>' overhang; however, homology directed repair is initiated by <NUM>'-><NUM>' end resection of DSBs, resulting in <NUM>' overhangs that are substrates for binding of RAD51 and that can interact further with the HDR machinery. Using nucleases that leave <NUM>' overhangs is expected to create DSBs that will more likely be repaired by HDR rather than NHEJ.

A number of strategies could be used to create such overhangs:.

Spo11 generates DSBs in meiotic cells, is required for synapsis, and remains covalently bound to DSBs after cleavage. It is believed that Mre11 exonuclease may process Spo11-bound DSBs to produce <NUM>' ends that are ideal substrates for HDR. One strategy for enhancing HDR then would be to fuse an engineered DNA binding domain (ZF, TALE, dCas9/gRNA) to Spo11 (in either N-, C-, or internal fusions) to DNA with appropriate spacing. This would be performed by targeting a pair of DBD-Spo11 monomers with appropriate spacing to create targeted DSBs with <NUM>' overhangs.

The sequence of human Spo11 isoform A is as follows:
<IMG>.

The sequence of human Spo11 isoform B is as follows:
<IMG>.

Thus, provided herein are Spo11-DBD fusion proteins with a DBD as described herein fused to the C terminus or N terminus of Spo11, optionally with an intervening linker sequence of <NUM>-<NUM> amino acids.

Recent evidence suggests that the chromatin context of a DSB may also influence repair pathway choice. For example, LEDGF, bound to H3K36me3, can recruit CtIP, a factor critical for end resection. Thus, provided herein are fusions of chromatin modifiers including, but not limited to SETD2, SRCAP, and SMARCAD1 to determine whether initiation of end resection and ultimately DNA repair outcomes can be biased in favor of HDR. Chromatin modifying proteins or domains may be fused directly to Cas9 or other DBDs, or localized via aforementioned dimerization/recruitment approaches.

It has been recently reported that nuclease-mediated gene targeting using protein-capped adenoviral donor vectors (<NPL>) results in precise repair with higher frequencies than with free-ended integration-defective lentiviral vectors (IDLV) or plasmid donors. This result was shown to depend on protein capping of the adenoviral donor DNA. In vitro studies in yeast have also shown that (<NPL>) the MRX-mediated resection of DNA is stimulated by the presence of protein blocks on the DNA ends. We envision the use of simple donor templates created by PCR using <NUM>' - or <NUM>'-biotinylated primers and associated with streptavidin, resulting in a protein-capped donor template.

The fusion proteins described herein can include any DNA Binding Domain (DBD) known in the art or engineered for a specific binding site. Exemplary DBDs include engineered or native TAL effector repeat arrays, engineered or native zinc fingers, modified variants (e.g., catalytically inactive) of homing meganucleases, modified variants (e.g., catalytically inactive) nucleases from the CRISPR-Cas system, chemical nucleases, and other native DBDs.

TAL effectors of plant pathogenic bacteria in the genus Xanthomonas play important roles in disease, or trigger defense, by binding host DNA and activating effector-specific host genes. Specificity depends on an effector-variable number of imperfect, typically ~<NUM>-<NUM> amino acid repeats. Polymorphisms are present primarily at repeat positions <NUM> and <NUM>, which are referred to herein as the repeat variable-diresidue (RVD). The RVDs of TAL effectors correspond to the nucleotides in their target sites in a direct, linear fashion, one RVD to one nucleotide, with some degeneracy and no apparent context dependence. In some embodiments, the polymorphic region that grants nucleotide specificity may be expressed as a triresidue or triplet.

Each DNA binding repeat can include a RVD that determines recognition of a base pair in the target DNA sequence, wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence. In some embodiments, the RVD can comprise one or more of: HA for recognizing C; ND for recognizing C; HI for recognizing C; HN for recognizing G; NA for recognizing G; SN for recognizing G or A; YG for recognizing T; and NK for recognizing G, and one or more of: HD for recognizing C; NG for recognizing T; NI for recognizing A; NN for recognizing G or A; NS for recognizing A or C or G or T; N* for recognizing C or T, wherein * represents a gap in the second position of the RVD; HG for recognizing T; H* for recognizing T, wherein * represents a gap in the second position of the RVD; and IG for recognizing T.

TALE proteins may be useful in research and biotechnology as targeted chimeric nucleases that can facilitate homologous recombination in genome engineering (e.g., to add or enhance traits useful for biofuels or biorenewables in plants). These proteins also may be useful as, for example, transcription factors, and especially for therapeutic applications requiring a very high level of specificity such as therapeutics against pathogens (e.g., viruses) as non-limiting examples.

Methods for generating engineered TALE arrays are known in the art, see, e.g., the fast ligation-based automatable solid-phase high-throughput (FLASH) system described in <CIT>, and <NPL>); as well as the methods described in <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>);<NPL>); <NPL>);<NPL>);<NPL>);<NPL>); <NPL>);<NPL>);<NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>);<NPL>); and <NPL>).

Zinc finger proteins are DNA-binding proteins that contain one or more zinc fingers, independently folded zinc-containing mini-domains, the structure of which is well known in the art and defined in, for example, <NPL>; <NPL>; <NPL>; and <NPL>. Crystal structures of the zinc finger protein Zif268 and its variants bound to DNA show a semi-conserved pattern of interactions, in which typically three amino acids from the alpha-helix of the zinc finger contact three adjacent base pairs or a "subsite" in the DNA (<NPL>; <NPL>). Thus, the crystal structure of Zif268 suggested that zinc finger DNA-binding domains might function in a modular manner with a one-to-one interaction between a zinc finger and a three-base-pair "subsite" in the DNA sequence. In naturally occurring zinc finger transcription factors, multiple zinc fingers are typically linked together in a tandem array to achieve sequence-specific recognition of a contiguous DNA sequence (<NPL>).

Multiple studies have shown that it is possible to artificially engineer the DNA binding characteristics of individual zinc fingers by randomizing the amino acids at the alpha-helical positions involved in DNA binding and using selection methodologies such as phage display to identify desired variants capable of binding to DNA target sites of interest (<NPL>; <NPL>; <NPL>; <NPL>). Such recombinant zinc finger proteins can be fused to functional domains, such as transcriptional activators, transcriptional repressors, methylation domains, and nucleases to regulate gene expression, alter DNA methylation, and introduce targeted alterations into genomes of model organisms, plants, and human cells (<NPL>; <NPL>; <NPL>).

Widespread adoption and large-scale use of zinc finger protein technology have been hindered by the continued lack of a robust, easy-to-use, and publicly available method for engineering zinc finger arrays. One existing approach, known as "modular assembly," advocates the simple joining together of pre-selected zinc finger modules into arrays (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>;<NPL>). Although straightforward enough to be practiced by any researcher, recent reports have demonstrated a high failure rate for this method, particularly in the context of zinc finger nucleases (<NPL>; <NPL>), a limitation that typically necessitates the construction and cell-based testing of very large numbers of zinc finger proteins for any given target gene (<NPL>).

Combinatorial selection-based methods that identify zinc finger arrays from randomized libraries have been shown to have higher success rates than modular assembly (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>). In preferred embodiments, the zinc finger arrays are described in, or are generated as described in, <CIT> and <CIT>. Additional suitable zinc finger DBDs are described in <CIT>, <CIT>, <CIT>, and <CIT> and <CIT>.

In some embodiments, a native DBD (e.g., a portion of a wild-type, non-engineered DNA binding protein that binds to a specific target sequence) can be used. For example, the DBD from a transcription factor, nuclease, histone, telomerase, or other DNA binding protein can be used. Typically DBDs include a structure that facilitates specific interaction with a target nucleic acid sequence; common DBD structures include helix-turn-helix; zinc finger; leucine zipper; winged helix; winged helix turn helix; helix-loop-helix; and hmg-box. The native DBD can be from any organism. See, e.g., <NPL>). The residues in a DNA binding protein that contact DNA, and thus form part of the DBD, can be determined empirically or predicted computationally, e.g., as described in<NPL>). A database of DNA binding proteins can be used to identify DNA binding proteins and DBDs for use in the present compositions and methods; see, e.g., <NPL>);<NPL>);<NPL>); <NPL>); <NPL>).

Where a native DBD is used in a fusion protein described herein, the catalytic domain is from a different protein.

Meganucleases are sequence-specific endonucleases originating from a variety of organisms such as bacteria, yeast, algae and plant organelles. Endogenous meganucleases have recognition sites of <NUM> to <NUM> base pairs; customized DNA binding sites with 18bp and 24bp-long meganuclease recognition sites have been described, and either can be used in the present methods and constructs. See, e.g., <NPL>); <NPL>);<NPL>); and <NPL>); <NPL>). In some embodiments, catalytically inactive versions of the homing meganucleases are used, e.g., a mutant of I-SceI, e.g., comprising the mutation D44S, wherein the catalytically active aspartate from the first LAGLIDADG motif is mutated to serine to make the enzyme inactive; N152K, reported to have ~ <NUM> % of the wt-activity; or the double variant D150C/N152K, which decreases the activity of the enzyme even further, e.g., as described in <NPL>; <NPL>; and <NPL>.

Catalytically inactive versions of the Cas9 nuclease can also be used as DBDs in the fusion proteins described herein; these fusion proteins are used in combination with a single guide RNA or a crRNA/tracrRNA pair for specificity. A number of bacteria express Cas9 protein variants. The Cas9 from Streptococcus pyogenes is presently the most commonly used; some of the other Cas9 proteins have high levels of sequence identity with the S. pyogenes Cas9 and use the same guide RNAs. Others are more diverse, use different gRNAs, and recognize different PAM sequences as well (the <NUM>-<NUM> nucleotide sequence specified by the protein which is adjacent to the sequence specified by the RNA). Chylinski et al. classified Cas9 proteins from a large group of bacteria (<NPL>), and a large number of Cas9 proteins are listed in supplementary <FIG> and supplementary table <NUM> thereof. The constructs and methods described herein can include the use of any of those Cas9 proteins, and their corresponding guide RNAs or other guide RNAs that are compatible. The Cas9 from Streptococcus thermophilus LMD-<NUM> CRISPR1 system has also been shown to function in human cells in <NPL>)). Additionally, Jinek et al. showed in vitro that Cas9 orthologs from S. thermophilus and L. innocua, (but not from N. meningitidis or C. jejuni, which likely use a different guide RNA), can be guided by a dual S. pyogenes gRNA to cleave target plasmid DNA, albeit with slightly decreased efficiency. These proteins are preferably mutated such that they retain their ability to be guided by the single guide RNA or a crRNA/tracrRNA pair and thus retain target specificity, but lack nuclease activity.

In some embodiments, the present system utilizes the Cas9 protein from S. pyogenes, either as encoded in bacteria or codon-optimized for expression in mammalian cells, containing mutations to render the nuclease portion of the protein catalytically inactive, e.g., mutations at D10, E762, H983, or D986; and at H840 or N863, e.g., at D10 and H840, e.g., D10A or D10N and H840A or H840N or H840Y; see, e.g., <NPL>; <NPL>).

DNA binding domains from the so-called "chemical nucleases,''(<NPL>)), e.g., triplex-forming oligonucleotides or peptide nucleic acids can also be utilized in the present compositions and methods; see, e.g.,<NPL>; <NPL>; <NPL>; <NPL>; or <NPL>.

The fusion proteins described herein can include any nuclease known in the art. Exemplary nucleases include engineered TALENs, zinc finger nucleases (ZFNs), homing meganucleases, nucleases from the CRISPR-Cas system, and other chemical nucleases. In some embodiments, a catalytically active nuclease domain is used, e.g., a Fok I cleavage domain. Some of the nuclease systems are described generally in <NPL>; <NPL>;.

Transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease (e.g., a Fok I cleavage domain) fused to a DNA-binding domain that can be easily engineered so that TALENs can target essentially any sequence (See, e.g., <NPL>)). Methods for generating engineered TALENs are known in the art, see, e.g., the fast ligation-based automatable solid-phase high-throughput (FLASH) system described in <CIT>, and <NPL>); as well as the methods described in <NPL>);<NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>);<NPL>); <NPL>); <NPL>); <NPL>); <NPL>);<NPL>); <NPL>); <NPL>); and <NPL>).

Zinc-finger nucleases (ZFNs) are composed of programmable, sequence-specific zinc finger DNA-binding modules (see above) linked to a nonspecific DNA cleavage domain, e.g., a Fok I cleavage domain. Methods for making and using ZFNs are known in the art, see, e.g., (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>). In some embodiments, the ZFNs are described in, or are generated as described in, <CIT> or <CIT>. Additional suitable ZFNs are described in <CIT>, <CIT>, <CIT>, and <CIT> and <CIT>.

As noted above, meganucleases are sequence-specific endonucleases originating from a variety of organisms such as bacteria, yeast, algae and plant organelles. A number of Meganucleases are known in the art, see, e.g., <CIT> (Meganuclease variants cleaving DNA target sequences of the TERT gene); <CIT>; <CIT> and <CIT> (<CIT> (I-CreI Meganuclease Variants with Modified Specificity).

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (<NPL>);<NPL>); <NPL>)) can serve as the basis for performing genome editing in bacteria, yeast and human cells, as well as in vivo in whole organisms such as fruit flies, zebrafish and mice (<NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>);<NPL>); <NPL>c);<NPL>);<NPL>)). The Cas nuclease, e.g., the Cas9 nuclease from S. pyogenes (hereafter simply Cas9), can be guided via base pair complementarity between the first <NUM>-<NUM> nucleotides of an engineered guide RNA (gRNA) and the complementary strand of a target genomic DNA sequence of interest that lies next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG (<NPL>); <NPL>);<NPL>); <NPL>); <NPL>); <NPL>); <NPL>c); <NPL>); <NPL>)). See also <NPL>; <NPL>, <CIT>; <CIT>; and <CIT>.

Chemical nucleases, e.g., triplex-forming oligonucleotides or peptide nucleic acids can also be utilized in the present compositions and methods; see above.

FokI is a type IIs restriction endonuclease that includes a DNA recognition domain and a catalytic (endonuclease) domain. The fusion proteins described herein can include all of FokI or just the catalytic endonuclease domain, e.g., amino acids <NUM>-<NUM> or <NUM>-<NUM> of GenBank Acc. No. AAA24927. <NUM>, e.g., as described in <CIT>,<NPL>); <NPL>), or a mutated form of FokI as described in <NPL>); <NPL>); or<NPL>). See also <NPL>.

An exemplary amino acid sequence of FokI is as follows:
<IMG>
<IMG>.

An exemplary nucleic acid sequence encoding FokI is as follows:
<IMG>.

In some embodiments, the FokI nuclease used herein is at least about <NUM>% identical SEQ ID NO:<NUM>, e.g., to amino acids <NUM>-<NUM> or <NUM>-<NUM> of SEQ ID NO:<NUM>. These variant nucleases must retain the ability to cleave DNA. In some embodiments, the nucleotide sequences are about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% identical to amino acids <NUM>-<NUM> or <NUM>-<NUM> of SEQ ID NO:<NUM>. In some embodiments, any differences from amino acids <NUM>-<NUM> or <NUM>-<NUM> of SEQ ID NO:<NUM> are in non-conserved regions.

To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least <NUM>% (in some embodiments, about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the length of the reference sequence is aligned). The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For purposes of the present application, the percent identity between two amino acid sequences is determined using the <NPL>) algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum <NUM> scoring matrix with a gap penalty of <NUM>, a gap extend penalty of <NUM>, and a frameshift gap penalty of <NUM>.

In order to use the fusion proteins described herein, it will generally be desirable to express them in a cell from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the fusion proteins can be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Suitable vectors include prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the fusion proteins. The nucleic acid encoding the fusion proteins can also be cloned into an expression vector, for expression in a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.

To obtain expression, a sequence encoding a fusion proteins is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in <NPL>); <NPL>); and <NPL>). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp. , and Salmonella (<NPL>). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the fusion proteins is to be expressed in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the guide RNA. In addition, a preferred promoter for expression of the fusion proteins can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-<NUM> system (see, e.g., <NPL>; <NPL>; <NPL>; <NPL>; and <NPL>).

In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the fusion proteins, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the fusion proteins, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.

Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-<NUM>, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

The vectors for expressing the fusion proteins can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SK promoters. These human promoters allow for expression of gRNAs in mammalian cells following plasmid transfection. Alternatively, a T7 promoter may be used, e.g., for in vitro transcription, and the RNA can be transcribed in vitro and purified.

Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the fusion proteins encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., <NPL>; <NPL>)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., <NPL>; <NPL>).

Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the fusion proteins.

The present invention includes the vectors and cells comprising the vectors, as well as cells expressing the fusion proteins described herein.

Claim 1:
A composition comprising a nuclease that induces DSBs only in specific phases of the cell cycle, the nuclease comprising a fusion protein comprising a cell-cycle regulated protein domain linked to an engineered nucleases, wherein the cell-cycle regulated protein domain is CtIP, Cdk2, Cyclin A1, Cyclin A2, Cyclin B1, or Gemini.