WO2019089623A1 - Fusion proteins for use in improving gene correction via homologous recombination - Google Patents

Abstract

Provided herein are fusion polypeptides each comprising a gene-editing nuclease enzyme such as CRISPR/Cas9 and a dominant-negative variant of p53 binding protein 1 (53BP1 DN variant). Such a fusion polypeptide can be used in gene editing to inhibit non- homologous end-joining (NHEJ) and enhancing homology-directed DNA repair.

Description

FUSION PROTEINS FOR USE IN IMPROVING GENE CORRECTION VIA

HOMOLOGOUS RECOMBINATION

CROSS-REFERENCE TO RELATED APPLICATIONS

5 This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/578,593, filed October 30, 2017, the contents of which are

incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

o Genome editing technologies have enabled a new paradigm to manipulate genome of host cells to achieve therapeutic effects, including correction of gene mutations associated with diseases and addition of therapeutic genes to desired sites of the genome. Genome editing technologies involve the use of endonucleases such as CRISPR/Cas9, zinc finger nuclease (ZFN), transcription activator-effector nucleases (TALENs) and meganuclease.5 Such endonucleases cleave DNAs at specific sites to create double strand breaks (DSBs), which would trigger endogenous cellular DNA repair systems.

There are two major cellular pathways to repair DSBs: homology-directed repair (HDR) and nonhomologous end-joining (NHEJ). The HDR pathway repairs the DSBs using a templated having homologous sequences as the damaged DNA, thereby allowing for o precise genome editing. By contrast, the NHEJ pathway repairs the DSBs by ligation of

DNA ends after they have been processed, thus resulting in imprecise small insertions and deletions. The NHEJ error prone repair occurs much more rapidly than HDR, and is therefore the far more predominant repair that can result in further mutations that are clinically undesirable.

5 Most cells, and especially hematopoietic stem cells, primarily utilize NHEJ to repair the genome from naturally occurring DNA breaks, which occur at a relatively high frequency. Hence, small molecules and decoys that have been used to inhibit NHEJ repair pathway in cells (global NHEJ inhibition) can be genotoxic and impair the natural DSB repair.

0 It is therefore of great interest to develop new strategies to inhibit NHEJ and/or

enhance HDR so as to achieve precise genome editing. SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the development of a number of dominant-negative p53 binding protein 1 variants (53BP1 DN variants or mutants), which can be recruited to DNA damage sites but cannot recruit other proteins of the NHEJ machinery. When fused with a gene-editing enzyme such as Cas9, the fusion proteins successfully inhibited NHEJ and increased HDR in gene editing, specifically only at the Cas9 nuclease cut site (site-specific NHEJ inhibition).

Accordingly, one aspect of the present disclosure features a fusion polypeptide, comprising a gene-editing nuclease enzyme and a dominant-negative variant of a p53 binding protein 1 (53BP1). The dominant-negative variant of 53BP1 is a truncated 53BP1, which may comprise the minimum focus forming region. In some embodiments, the dominant- negative variant of 53BP1 comprises (a) deletion in a docking domain, (b) a deletion of a BRCT domain, or (c) both (a) and (b). In some embodiments, the dominant negative variant of 53BP1 comprises (a) a deletion of region 1-1231 of SEQ ID NO: l or a portion thereof, (b) a deletion of region 1711-1972 of SEQ ID NO: 1 or a portion thereof, or (c) both (a) and (b). In some examples, the gene-editing nuclease enzyme is covalently linked directly to the dominant-negative variant of 53BP1 in the fusion polypeptide disclosed herein. Alternatively the gene-editing nuclease enzyme is linked to the dominant-negative variant of 53BP1 via a peptide linker.

The gene-editing nuclease enzyme in any of the fusion proteins disclosed herein may be any site-specific nuclease, such as a Cas9 enzyme (e.g., from a suitable bacterium), a Casl2 enzyme, a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN) or meganuclease such as a homing endonuclease. In some examples, the gene- editing nuclease enzyme is a Cas9 enzyme, which may comprise the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5 (which are encoded by the 5' portion of the nucleotide sequences (upstream to the linker sequence) shown in SEQ ID NO: 7 and SEQ ID NO: 9, respectively.

In some embodiments, the dominant-negative variant of 53BP1 comprises region 1480-1644 of SEQ ID NO: l. For example, the dominant-negative variant of 53BP1 comprises region 1231-1644 of SEQ ID NO: l, region 1231-1711 of SEQ ID NO: l, or 1480- 1711 of SEQ ID NO: l. In some examples, the dominant-negative variant of 53BP1 consists of 1231-1711 of SEQ ID NO: 1, 1231-1644 of SEQ ID NO: 1, or 1480-1711 of SEQ ID NO: 1. In specific examples, the the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, which are encoded by the nucleotide sequences shown in Figures 8 and 9 respectively.

In other aspects, provided herein is a nucleic acid, comprising a nucleotide sequence 5 coding for any of the fusion polypeptides disclosed herein. Also provided herein is a vector that comprises such a nucleic acid. In some instances, the nucleotide sequence coding for the fusion polypeptide in the vector is in operable linkage to a promoter, for example, a mammalian promoter. In some examples, the vector can be a viral vector such as an integration defective viral vector, e.g. , a retroviral vector, an adenoviral vector, an adeno- o associated viral vector, or a hybrid vector. In specific examples, the viral vector can be a retroviral vector, for example, a lentiviral vector.

In some embodiments, any of the vectors described herein may further comprise a nucleotide sequence coding for a guide RNA, and/or a sequence that serves as a template for homologous recombination, or both.

5 In yet another aspect, the present disclosure features a method for enhancing

homology directed DNA repair (HDR) in gene editing of a cell (e.g. , a mammalian cell such as a human cell). The method comprises introducing into a cell any of the fusion

polypeptides disclosed herein or a vector that comprises a nucleotide sequence coding for the fusion polypeptide, and optionally a guide RNA targeting a gene of interest. The fusion o polypeptide may be introduced into the cell in the form of a protein, an RNA or a DNA that encode the fusion polypeptide. In some instances, the guide RNA can be a single guide RNA. In some embodiments, the fusion polypeptide and the gRNA can be introduced into the cell by delivering a vector that expresses both the fusion polypeptide and the guide RNA into the cell.

5 In some embodiments, the method may further comprise introducing into the cell a donor template nucleic acid, which comprises homologous arms flanking a cleavage site in the gene of interest directed by the guide RNA. In some examples, the fusion polypeptide can be delivered into the cells in a ribonucleoprotein complex (RNP) form, which may further comprise a guide RNA, modified guide RNA, and/or a donor template nucleic acid as0 disclosed herein.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1 includes diagrams showing identification of the homology directed DNA repair (HDR)-enhancer fragment of p53 binding protein 1 (53BP1). A: Schematic diagram of 53BP1 protein showing (a) the minimal focus forming region (FFR) including the

oligomerization domain (OD), a glycine- arginine rich (GAR) motif, a tandem Tudor domain (TD), and the ubiquitin-dependent recruitment (UDR) motif; (b) the different truncated 53BP1 proteins (DN1, DNls, DN2, DN3, and DN4) that were tested for their ability to compete with the endogenous 53BP1 and recruit HDR proteins and not NHEJ proteins to the DNA DSB. The amino acid sequence regions are indicated for each 53BP1 truncated fragment. B: A Western blot photo showing relative expression levels of the different truncated human 53BP1 proteins (DN1, DN1S, DN2, DN3 or DN4, which was HA-tagged) which were analyzed by western blot using anti-HA antibodies and control anti-actin antibodies in HeLa cells transduced with empty vector (Mock) or lentiviral vectors encoding the HA-tagged truncated 53BP1 proteins. Each arm had equal lentiviral transduction rates in HeLa cells. C: A photo showing the relative expression levels of DN2, DN3 and DN4 as shown B, at higher exposure to show DN2 levels. D: a chart showing quantification of the numbers of the cells with HA+ (DN1S) foci or HA+ endogenous 53BP1+ (Co-localization) foci or endogenous 53BP1+ only foci. E: A chart showing quantification of the numbers of the cells with >3 RIF- 1 foci in control cells or irradiated cells with or without the presence of DN1/DN1S. F: A chart showing quantification of the numbers of the cells with >3 BRCA-1 foci in control cells or irradiated cells with or without the presence of DN1/DN1S. G: A chart showing quantification of the numbers of the cells with >3 γΗ2ΑΧ foci in control cells or irradiated cells with or without the presence of DN1/DN1S. The cumulative data in Figure 1 shows that DN1 and DN1S versions of 53BP1 are expressed at high levels and function as a dominant negative (DN) protein: like the endogenous 53BP1 are both recruited to DSBs (at γ- H2AX foci), where they either co-localize or displace endogenous 53BP1, reduce binding of the downstream NHEJ recruiter protein RIF-1, and increase binding of the HDR protein BRCA-1.

Figure 2 includes diagrams showing Cas9-DN1S fusion protein locally inhibits NHEJ, thereby reducing cellular toxicity that is normally seen with global NHEJ inhibition. A: a schematic diagram showing the different truncated 53BP1 proteins (DNl, DNIS, DN2 and DN2L) fused with Cas9. B: a photo showing the relative expression levels of the different Cas9 fusion proteins as analyzed by western blot using anti-FLAG antibodies and control anti-actin antibodies in HeLa cells transfected transduced with plasmids encoding an FLAG-tagged Cas9 fusion protein and mCherry fluorophore. Each arm was sorted for mCherry+ cells. The blot shows that DNl, DNIS, DN2 and DN2L fusions with Cas9 result in the expected size stable protein expression. C: A representative photo showing

representative immunofluorescence (IF) images showing HA tagged DNIS or dCas9- DNlS/gRNA (red fluorescence) and endogenous 53BP1 (green fluorescence) recruitment to the DNA break site. Nuclie are labeled with DAPI (blue). D. A chart showing quantification of the numbers of the cells with >1 HA+ (DNIS) foci. E: A chart showing quantification of the number of the cells with >3 53BP1 foci in mock (cells transduced with empty vector), DNIS (cells transduced with lentiviral vectors expressing DNIS fragment), dCas9 (cells transduced with lentiviral vectors expressing dCas9), dCas9-DNlS (cells transduced with lentiviral vectors expressing dCas9-DNlS) and dCas9-DNlS/gRNA (cells transduced with lentiviral vectors expressing dCas9-DNlS/gRNA). F: Ionizing radiation (IR) sensitizes cells to apoptosis and death if cellular NHEJ -based repair pathways are compromised. This was tested using a colony forming assay. A chart showing viability of HeLa cells treated with ionizing radiation (IR) at the indicated doses after expressing the fusion protein and gRNA, and global NHEJ inhibition (with shRNA to 53BP1 or NU7441) or their appropriate controls, as indicated. The viable colonies were determined by crystal violet staining. IR treatment resulted in a significant loss of viability with global NHEJ inhibition (using sh53BPl or NU7441), and a dose-dependent decrease in colony formation when compared with the controls. The data are presented as mean + SEM of three tests, *P<0.05, **P<0.01. G: A chart showing viability of cells treated with IR at the indicated doses and expressing a catalytically inactive form of Cas9 (dCas9) fused to DNIS (dCas9-DNlS with gRNA). The cell viability was determined by crystal violet staining. IR treatment resulted in no significant dose dependent or dCas9-DNlS/gRNA dependent decrease in colony formation when compared with the controls. dCas9-DNlS/gRNA transfected HeLa cells show that despite continuous presence of the catalytically dead Cas9-DN1S fusion protein (tethered to the Cas9 specific sites by the gRNA), cells were not sensitized to IR. These data show that a dead Cas9 5 is not dragged by the DN1S to IR induced DSB and cause toxicity.

The cumulative data in Figure 2 shows that fusion of the DN 53BP1 fragment to Cas9 results in stable fusion protein expression. Unlike the DN 53BP1 alone, which localizes to DSBs, the Cas9-DN constructs form very limited foci.

Figure 3 includes diagrams illustrating the Traffic Light Reporter (TLR) system. A: a o diagram showing construction of the TLR system in exemplary host cells, the 293T cells. B:

a diagram showing the use of CRISPR/Cas9 to the TLR locus in 293T cells to test the relative efficiency of NHEJ and HDR. Nucleotide sequence of AAVS l-1 (top): SEQ ID NO: 10. Nucleotide sequence of AAVS 1-2 (bottom): SEQ ID NO: 11. C. Bar diagram showing the relative frequencies of HDR and NHEJ induced by different Cas9 fusion proteins (DN1,5 DN1S, DN2, DN2L) as compared to those induced by Cas9 using the TLR system in 293T cells. DN1 or DN1S significantly increase HDR, and the HDR/NHEJ ratio as compared to Cas9 alone. D. Relative frequencies of HDR induced by different SpCas9-DNIS fusion proteins with different tags and linkers were compared to those induced by Cas9 using TLR system in 293T cells. The DN1S can be fused to Cas9 using a variety of linkers and tags, with o the same effect on increased HDR, compared to the respective Cas9 controls.

Figure 4 includes diagrams showing HDR stimulation by the Cas9-DN1S fusion protein, which takes place at different target genes/loci in multiple cell lines as indicated. A: Cas9 derived from Streptococcus pyogenes (SpCas9) was used as indicated. A chart showing relative frequencies of HDR induced by different SpCas9 fusion proteins as indicated, which 5 were compared to those induced by Cas9. 293T cells were targeted either at the AAVS 1 locus (left panel) or the LM02 locus (middle panel) using the spCas9 plasmid and a GFP donor homology template. Relative frequencies of GFP+ (HDR) cells using either spCas9 or spCas9-DNlS fusion protein and the percentage of GFP+ cells shown. GFP was targeted in frame into the CD45 gene locus of K562 cells (right panel). B: Cas9 derived from

0 Staphylococcus aureus (SaCas9), or its fusion with DN1S (SaCas9-DN) shows a highly

significant reduction of NHEJ and increase in HDR in EBV transformed primary human B cells (LCL) at the CD45 locus (left panel) or the AAVS 1 locus (middle panel). Similar extremely high efficiency HDR, reaching >90% was observed in the K562 hematopoietic cells targeted with the SaCas9-DN. Red bars denote NHEJ frequency and Green bars denote HDR. For hematopoietic cells, SaCas9 and SaCas9-DN proteins were complexed with gRNA to the indicated loci and transfected as a ribonucleoprotein complex (RNP). C: A bar plot showing the gene editing efficiency (NHEJ or HDR) of SaCas9 or SaCas9-DNlS using the CD45 reporter system in K562 cells. When the CD45 locus was targeted in K562 cells using the SaCas9-DN, we did not observe increased HDR over SaCas9 alone. However, there was a very highly significant reduction in NHEJ/error prone repair.

The cumulative data from Figure 4 shows that the fusion of the dominant negative 53BP1 fragment significantly increases HDR and reduces NHEJ at different target genes, in multiple cell lines, using different Cas9 nucleases. Occasionally, if HDR is not increased, a highly significant decrease in NHEJ occurs at the nuclease induced DSB.

Figure 5 includes diagrams showing targeting CD 18 at the AAVS 1 locus in B lymphocytes derived from a patient with Leukocyte Adhesion Deficiency (which results from defects in the CD 18 gene) showed higher levels and quality of HDR. A: A diagram showing epresentative flow cytometry plots of EBV immortalized primary B cells from a patient with Leukocyte Adhesion Defect (LAD) transfected with the indicated conditions for targeted integration of CD18 at the AAVS 1 locus. LAD results from lack of expression of the CD18 integrin (adhesion molecule). The fractions of unedited cells (black) or HDR+ cells (green at the right gated portion; percentage indicated) are shown with appropriate controls. Here,

LAD B cells were transfected either with SaCas9/gRNA RNP, or SaCas9-DN RNP along with a homology donor template (DT) carrying the CD 18 gene was embedded in the AAV-6 virus for efficient delivery. No RNP and no DT controls are indicated. The gRNA was designed to target the AAVS 1 locus. The flow cytometry plots show that not only was a higher percentage of HDR (GFP+ cells) were seen with SaCas9-DN, but the HDR population showed much brighter GFP fluorescence. B: A bar diagram showing the quantification of the HDR efficiency and NHEJ efficiency of the SaCas9 or SaCas9-DNlS by flow cytometry (green) and TIDE assay (red), respectively in LAD B cells with AAVS 1 CD 18 donor, showing a highly significant increase in HDR and proportionate decrease in NHEJ in EBV immortalized LAD B lymphocytes. C. The GFP+ HDR cells could be separated into a GFP^bright (bi-allelic HDR) and GFP^™ (mono-allelic HDR), as confirmed by sorting this population and subjecting the bright and dim cells to PCR for HDR. The relative frequencies of mono-allelic and bi-allelic HDR frequency detected by MFI of CD 18 staining shows that the SaCas9-DN fusion protein highly significantly improves the HDR on both alleles. A bi- allelic correction would result in perfect correction of the CD 18 expression to normal levels.

Figure 6 is a bar plot showing the NHEJ editing efficiency of Cas9 or Cas9-DN1S at the top four off target sites of AAVS 1 gRNA in EB V immortalized B cells from a LAD patient. The Cas9-DN1S fusion decreases or does not increase the off target cutting.

DETAILED DESCRIPTION OF THE INVENTION

Double-strand breaks (DSBs) are very common both in quiescent cells and when cells undergo replication. Normally, cells (especially hematopoietic stem cells) use nonhomologous end joining (NHEJ) to repair DNA double strand breaks. Hence, global blockade of the NHEJ pathway using small molecule inhibitors to 53BP1, DNA-PK, Ku70/80, ligase 4, etc. are toxic to hematopoietic stem cells (resulting in apoptosis due to unrepaired DSBs) or can potentially result in deleterious, even cancer-causing mutations.

Upon DNA damage, ATM phosphorylates 53BP1. 53BP1 is the first protein to be recruited to DNA damage sites, which then recruits the RIF-1/PTIP protein complex to recruit the other NHEJ proteins to the damage sites to carry out NHEJ repair. We discovered that dominant negative variants of 53BP1, when fused to CRISPR/Cas9 inhibited NHEJ and enhanced HDR in gene editing as observed in multiple cell lines and at various target gene sites. Accordingly, described herein are fusion proteins containing gene editing nucleases such as SpCas9 or SaCas9 (or other site-specific nucleases such as Casl2 (Cpfl) or ZFN) and a dominant negative mutant of the 53BP1 protein, which can be recruited to the DNA DSB sites but unable to recruit other NHEJ proteins. Such fusion proteins can be used to inhibit at sites where the gene editing nuclease creates a DSB, thereby inhibiting NHEJ and increase homology directed repair.

Unexpected, the studies provided herein showed that DN 53BP1 alone, while recruited to DSB, is toxic to cells as it interferes with naturally occurring NHEJ repair of cells. Further, it is reported here that DN 53BP1 alone competes with endogenous 53BP1 and displaces endogenous 53BP1 at a high enough level to result in toxicity. Moreover, it was discovered that fusing DN 53BP1 fragment to Cas9 not only decreases NHEJ only at the Cas9 cut site, but promotes HDR in multiple human cell types and loci. Fusion Polypeptides Containing Dominant-Negative 53BP1 Variants and Gene- Editing Enzymes

One aspect of the present disclosure relates to fusion polypeptides each comprising a gene-editing nuclease enzyme and a dominant- negative 53BP1 variant. As used herein, a fusion polypeptide refers to a polypeptide comprising at least two fragments derived from different parent proteins, for example, one fragment from a gene-editing nuclease enzyme and one fragment from a 53BP1 protein. In the fusion polypeptides described herein, the gene-editing nuclease enzyme can be linked directly to the 53BP1 DN variant. Alternatively, the gene-editing nuclease enzyme can be linked to the 53BP1 DN variant through a peptide linker, for example, a TGS linker (see below) and an XTEN linker. In some instances, the gene-editing nuclease enzyme is located at the N-terminus of the fusion polypeptide. In other instances, the 53BP1 DN variant is located at the N-terminus of the fusion polypeptide.

(A ) 53BP1 Dominant-Negative Variants

Tumor suppressor p53-binding protein 1 (53BP1) is a protein that plays an essential role in DNA damage repair. In humans, 53BP1 is encoded by the TP53BP1 gene. 53BP1 binds to the DNA-binding domain of p53 and enhances p53-mediated transcriptional activation. 53BP1 plays multiple roles in the DNA damage response, including promoting checkpoint signaling following DNA damage, acting as a scaffold for recruitment of DNA damage response proteins to damaged chromatin, and promoting NHEJ pathways by limiting end resection following a double- strand break.

53BP1 proteins of various species have been well characterized. Information of one exemplary human 53BP1 can be found under UniProtKB-Q12888. Exemplary amino acid sequence is provided below (SEQ ID NO: l).

10 2 0 30 4 0 50

MDPTGSQLDS DFSQQDTPCL I I ED SQPE SQ VLEDD SGSHF SMLSRHLPNL

60 7 0 80 90 100

QTHKENPVLD VVSNPEQTAG EERGDGNS GF NEHLKENKVA DPVD S SNLDT

1 10 12 0 130 14 0 150

CGS I SQVI EQ LPQPNRT S SV LGMSVE SAPA VEEEKGEELE QKEKEKEEDT

1 60 17 0 1 80 1 90 200

SGNTTHSLGA EDTAS SQLGF GVLELSQSQD VEENTVPYEV DKEQLQSVTT

2 10 22 0 230 24 0 250

NS GYTRLSDV DANTAIKHEE QSNED I P IAE QS SKD IPVTA QP SKDVHWK 260 270 280 290 300

EQNPPPARSE DMPFSPKASV AAMEAKEQLS AQELMESGLQ IQKSPEPEVL

310 320 330 340 350

STQEDLFDQS NKTVSSDGCS TPSREEGGCS LASTPATTLH LLQLSGQRSL

360 370 380 390 400

VQDSLSTNSS DLVAPSPDAF RSTPFIVPSS PTEQEGRQDK PMDTSVLSEE

410 420 430 440 450

GGEPFQKKLQ SGEPVELENP PLLPESTVSP QASTPISQST PVFPPGSLPI

460 470 480 490 500

PSQPQFSHDI FIPSPSLEEQ SNDGKKDGDM HSSSLTVECS KTSEIEPKNS

510 520 530 540 550

PEDLGLSLTG DSCKLMLSTS EYSQSPKMES LSSHRIDEDG ENTQIEDTEP

560 570 580 590 600

MSPVLNSKFV PAENDSILMN PAQDGEVQLS QNDDKTKGDD TDTRDDISIL

610 620 630 640 650

ATGCKGREET VAEDVCIDLT CDSGSQAVPS PATRSEALSS VLDQEEAMEI

660 670 680 690 700

KEHHPEEGSS GSEVEEIPET PCESQGEELK EENMESVPLH LSLTETQSQG

710 720 730 740 750

LCLQKEMPKK ECSEAMEVET SVISIDSPQK LAILDQELEH KEQEAWEEAT

760 770 780 790 800

SEDSSVVIVD VKEPSPRVDV SCEPLEGVEK CSDSQSWEDI APEIEPCAEN

810 820 830 840 850

RLDTKEEKSV EYEGDLKSGT AETEPVEQDS SQPSLPLVRA DDPLRLDQEL

860 870 880 890 900

QQPQTQEKTS NSLTEDSKMA NAKQLSSDAE AQKLGKPSAH ASQSFCESSS

910 920 930 940 950

ETPFHFTLPK EGDIIPPLTG ATPPLIGHLK LEPKRHSTP I GISNYPESTI

960 970 980 990 1000

ATSDVMSESM VETHDPILGS GKGDSGAAPD VDDKLCLRMK LVSPETEASE

1010 1020 1030 1040 1050

ESLQFNLEKP ATGERKNGST AVAESVASPQ KTMSVLSCIC EARQENEARS

1060 1070 1080 1090 1100

EDPPTTPIRG NLLHFPSSQG EEEKEKLEGD HTIRQSQQPM KPISPVKDPV

1110 1120 1130 1140 1150

SPASQKMVIQ GPSSPQGEAM VTDVLEDQKE GRSTNKENPS KALIERPSQN

1160 1170 1180 1190 1200

NIGIQTMECS LRVPETVSAA TQTIKNVCEQ GTSTVDQNFG KQDATVQTER

1210 1220 1230 1240 1250

GSGEKPVSAP GDDTESLHSQ GEEEFDMPQP PHGHVLHRHM RTIREVRTLV

1260 1270 1280 1290 1300

TRVITDVYYV DGTEVERKVT EETEEP IVEC QECETEVSPS QTGGSSGDLG

1310 1320 1330 1340 1350

DISSFSSKAS SLHRTSSGTS LSAMHSSGSS GKGAGPLRGK TSGTEPADFA

1360 1370 1380 1390 1400

LPSSRGGPGK LSPRKGVSQT GTPVCEEDGD AGLGIRQGGK APVTPRGRGR

1410 1420 1430 1440 1450

RGRPPSRTTG TRETAVPGPL GIEDISPNLS PDDKSFSRVV PRVPDSTRRT

1460 1470 1480 1490 1500

DVGAGALRRS DSPEIPFQAA AGPSDGLDAS SPGNSFVGLR WAKWSSNGY

1510 1520 1530 1540 1550

FYSGKITRDV GAGKYKLLFD DGYECDVLGK DILLCDPIPL DTEVTALSED 1560 1570 1580 1590 1600

EYFSAGWKG HRKESGELYY SIEKEGQRKW YKRMAVILSL EQGNRLREQY

1610 1620 1630 1640 1650

GLGPYEAVTP LTKAADI SLD NLVEGKRKRR SNVSSPATPT ASSSSSTTPT

1660 1670 1680 1690 1700

RKITESPRAS MGVLSGKRKL ITSEEERSPA KRGRKSATVK PGAVGAGEFV

1710 1720 1730 1740 1750

SPCESGDNTG EPSALEEQRG PLPLNKTLFL GYAFLLTMAT TSDKLASRSK

1760 1770 1780 1790 1800

LPDGPTGSSE EEEEFLEIPP FNKQYTESQL RAGAGYILED FNEAQCNTAY

1810 1820 1830 1840 1850

QCLLIADQHC RTRKYFLCLA SGIPCVSHVW VHDSCHANQL QNYRNYLLPA

1860 1870 1880 1890 1900

GYSLEEQRIL DWQPRENPFQ NLKVLLVSDQ QQNFLELWSE ILMTGGAASV

1910 1920 1930 1940 1950

KQHHSSAHNK DIALGVFDVV VTDPSCPASV LKCAEALQLP WSQEWVIQC

1960 1970

LIVGERIGFK QHPKYKHDYV SH

As illustrated in Figure 1 , panel A, an example 53BP1 includes 1972 amino acids. The N-terminal portion (1- 1231) contains domains for interacting with other proteins of cellular DNA repair machinery (docking domains). The C-terminal portion (1711- 1972) contains two breast cancer susceptibility gene 1 (BRCT) motifs, which are common motifs presented in several proteins involved in DNA repair and/or DNA damage- signaling pathways. Rappold et al., J. Cell Biol. 2001, 153(3):613-620. In between the docking domains and the BRCT domains is the minimal focus forming region (FFR), which may include residues 1231- 1711 of SEQ ID NO: l . The FFR region includes an

oligomerization domain (OD), a glycine- arginine rich (GAR) motif, a tandem Tudor domain (TD), and an ubiquitin-dependent recruitment (UDR) motif.

The dominant-negative variant of p53BPl protein as disclosed herein can comprise a fragment of a wild-type 53BP1 from a suitable species (e.g. , a mammal such as a human). An exemplary human 53BP1 is provided above. 53BP1 proteins from other species are well known in the art and their sequences can be retrieved from publically available gene database, for example, using SEQ ID NO: l as a search query. A dominant- negative variant of a 53BP1 protein refers to a mutant of the wild-type 53BP1 protein and adversely affects the normal bioactivity of the wild-type counterpart within the same cells. In some instances, the dominant-negative variant disclosed herein can maintain

substantially similar binding activity to a DNA damage site (e.g. , a DSB site) but has little or no activity in recruiting other protein components of the NHEJ repair machinery. Without being bound by theory, such a dominant-negative variant can compete against wild-type 53BP1 from binding to the DSB site but cannot recruit other NHEJ repair proteins, thereby inhibiting the NHEJ repair function of the wild-type 53BP1 counterpart in the same cells. When fused to a site specific nuclease (e.g. , Cas9), the dominant- negative variant of a 53BP1 protein may only restrict NHEJ at the specific site where the site-specific nuclease induces a DSB, and would not inhibit normal cellular NHEJ.

The 53BP1 dominant- negative (53BP1 DN) variants described herein may be a truncated version of a wild-type 53BP1, in which one or more of the docketing domains and/or one or more of the BRCT domains are deleted. A docketing domain in a 53BP1 protein is a functional domain, usually located in the N-terminal portion of the protein (e.g. , residues 1-1230 of SEQ ID NO: l), that recruits RIF- 1, PTIP and the rest of the proteins involved in NHEJ. For example, the 53BP1 DN variant may have a deletion of the fragment corresponding to residues 1- 1230 of SEQ ID NO: l (containing docking domains) or a portion thereof. Alternatively or in addition, the 53BP1 DN variant may have a deletion of the fragment corresponding to residues 1722 to 1972 of SEQ ID NO: l (containing BRCT domains) or a portion thereof. The 53BP1 DN variants disclosed herein may contain the minimal focus forming region (e.g. , residues 1231-1711 of SEQ ID NO: l) or a portion thereof, which binds DSB sites.

In some examples, the 53BP1 DN variant has the complete fragment

corresponding to residues 1- 1230 of SEQ ID NO: 1 deleted or has the complete fragment corresponding to residues 1722 to 1972 of SEQ ID NO: l deleted. In specific examples, the 53BP1 DN variant has both fragments corresponding to 1-1230 of SEQ ID NO: l and residues 1722-1972 of SEQ ID NO: l deleted.

In some embodiments, the 53BP1 DN variant disclosed herein contains one or more functional domains within the minimal focus forming region illustrated in Figure 1, panel A. For example, the 53BP1 DN variant may contain one or more of the OD domain, the GAR) motif, the TD domain, and the UDR) motif. In some examples, the 53BP1 DN variant contains the fragment corresponding to 1231-1711 of SEQ ID NO: l or a fragment thereof. Specific examples of 53BP1 DN variants are provided in Figure 1, panel A, including DN-53BP1 #1 (consisting of the fragment corresponding to residues 1231- 1711 of SEQ ID NO: l), DN-53BP1 #1S (consisting of the fragment corresponding to residues 1231-1644 of SEQ ID NO: l), DN-53BP1 #2 (containing the fragment corresponding to residues 1231-1644 of SEQ ID NO: l with a linker replacing the fragment of 1277-1480); DN-53BP1 #3 (consisting of the fragment corresponding to residues 1480- 1644 of SEQ ID NO: l), and DN-53BP1 #4 (consisting of the fragment corresponding to residues 1480- 1711 of SEQ ID NO: l).

The amino acid sequence of 53BP1 DN1S and its encoding nucleotide sequence, as an example, is provided below:

PHGHVLHRHMRT IREVRTLVTRVI TDVYYVDGTEVERKVTEETEEP IVECQECETEVSP SQTGGS S G DLGD I S SF S SKAS S LHRT S S GT SLSAMHS S GS SGKGAGPLRGKT SGTEPADFALP S SRGGPGKLSPR KGVSQTGTPVCEEDGDAGLGIRQGGKAPVTPRGRGRRGRPP SRTTGTRETAVPGPLGI ED I SPNLSP DDKSFSRVVPRVPD STRRTDVGAGALRRSD SPE I PFQAAAGP SDGLDAS SP GNSFVGLRVVAKWS SN GYFYSGKI TRDVGAGKYKLLFDDGYECDVLGKD I LLCDP I PLDTEVTALSEDEYFSAGWKGHRKE S GELYYS IEKEGQRKWYKRMAVI LS LEQGNRLREQYGLGPYEAVTPLTKAAD I SLDNLVEGKRKRRSN VS SPATPTAS S S ( SEQ I D NO : 2 ) CCACATGGCCATGTCTTACATCGTCACATGAGAACAATCCGGGAAGTACGCACACTTGTCACTCGTGT CATTACAGATGTGTATTATGTGGATGGAACAGAAGTAGAAAGAAAAGTAACTGAGGAGACTGAAGAGC CAATTGTAGAGTGTCAGGAGTGTGAAACTGAAGTTTCCCCTTCACAGACTGGGGGCTCCTCAGGTGAC CTGGGGGATATCAGCTCCTTCTCCTCCAAGGCATCCAGCTTACACCGCACATCAAGTGGGACAAGTCT CTCAGCTATGCACAGCAGTGGAAGCTCAGGGAAAGGAGCCGGACCACTCAGAGGGAAAACCAGCGGGA CAGAACCCGCAGATTTTGCCTTACCCAGCTCCCGAGGAGGCCCAGGAAAACTGAGTCCTAGAAAAGGG GTCAGTCAGACAGGGACGCCAGTGTGTGAGGAGGATGGTGATGCAGGCCTTGGCATCAGACAGGGAGG GAAGGCTCCAGTCACGCCTCGTGGGCGTGGGCGAAGGGGCCGCCCACCTTCTCGGACCACTGGAACCA GAGAAACAGCTGTGCCTGGCCCCTTGGGCATAGAGGACATTTCACCTAACTTGTCACCAGATGATAAA TCCTTCAGCCGTGTCGTGCCCCGAGTGCCAGACTCCACCAGACGAACAGATGTGGGTGCTGGTGCTTT GCGTCGTAGTGACTCTCCAGAAATTCCTTTCCAGGCTGCTGCTGGCCCTTCTGATGGCTTAGATGCCT CCTCTCCAGGAAATAGCTTTGTAGGGCTCCGTGTTGTAGCCAAGTGGTCATCCAATGGCTACTTTTAC TCTGGGAAAATCACACGAGATGTCGGAGCTGGGAAGTATAAATTGCTCTTTGATGATGGGTACGAATG TGATGTGTTGGGCAAAGACATTCTGTTATGTGACCCCATCCCGCTGGACACTGAAGTGACGGCCCTCT CGGAGGATGAGTATTTCAGTGCAGGAGTGGTGAAAGGACATAGGAAGGAGTCTGGGGAACTGTACTAC AGCATTGAAAAAGAAGGCCAAAGAAAGTGGTATAAGCGAATGGCTGTCATCCTGTCCTTGGAGCAAGG AAACAGACTGAGAGAGCAGTATGGGCTTGGCCCCTATGAAGCAGTAACACCTCTTACAAAGGCAGCAG ATATCAGCTTAGACAATTTGGTGGAAGGGAAGCGGAAACGGAGATCTAACGTCAGCTCCCCAGCCACC CCTACTGCCTCCTCGAGC ( SEQ I D NO : 3 ) Any of the 53BP1 DN variants disclosed herein may contain fragments from a native 53BP1 protein from any suitable species (e.g. , human, monkey, chimpanzee, mouse, rat, pig, etc.). Alternatively, it may contain a functional variant of the fragment from the native counterpart. A functional variant would maintain substantially similar bioactivity of the functional domains contained in the fragment of the native counterpart and share a high amino acid sequence homology with the native counterpart (e.g. , at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or above). The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403- 10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequencfes homologous to the protein

5 molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In some instances, a functional variant may contain conservative amino acid o residue substitutions relative to the native counterpart. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such

5 methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; o (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Any of the 53BP1 DN variants disclosed herein may have a length of up to 200- amino acid, up to 300-amino acid, 400-amino acid, up to 450-amino acid, up to 500-amino acid, up to 550-amino acid, up to 600-amino acid, or up to 700-amino acid. 5 (B) Gene-Editing Nuclease Enzyme

Any of the nucleases used in commonly known gene-editing methods can be used in making the fusion polypeptides disclosed herein. Genome editing methods are generally classified based on the type of endonuclease that is involved in generating double stranded breaks in the target nucleic acid. In some embodiments, the gene-editing0 nuclease enzyme disclosed herein is an RNA-guided endonuclease, which cleaves DNA at a site specific to a guide RNA. Exemplary gene-editing nuclease enzymes include, but are not limited to, zinc finger nucleases (ZFN), transcription activator-like effector-based nuclease (TALEN), meganucleases, and Cas9 or variants thereof (e.g. , Cas l2) for use in the CRISPR/Cas systems.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can 5 be engineered to target specific desired DNA sequences and this enables zinc-finger

nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.

Transcription activator-like effector nucleases (TALEN) are restriction enzymes o that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. TALEs can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing. Exemplary TALEN nucleases can be found at GenBank Accession5 No. AKB90849 or GenBank Accession No. AKB90848.

Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR/Cas9 that can be used to edit genes within organisms. This type of gene editing process has a wide variety of applications including use as a basic biology research tool, development of biotechnology products, and potentially to treat diseases. o Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double- stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. Exemplary meganucleases for use in gene editing include homing endonucleases. Meganucleases can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence 5 through protein engineering, the targeted sequence can be changed.

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme used in the CRISPR technology for gene editing. In some embodiments, the Cas9 enzyme can be from Streptococcus pyogenes. Cas9 proteins have been routinely used as a genome engineering tool to induce site-directed double strand breaks in DNA. These0 breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. When fused with the 53BP1 DN variants disclosed herein, the resultant fusion polypeptides can be used in CRISPR systems to inhibit NHEJ and enhance repair via homologous recombination. Exemplary Cas9 proteins for use in the present disclosure includes those encoded by the nucleotide sequences shown in Figures 8 and 9.

5 In some embodiments, the Cas endonuclease is a Cas9 enzyme or variant thereof. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus, or Treponema denticola. In some embodiments, the nucleotide sequence encoding the Cas endonuclease may be codon optimized for expression in a host cell. In some embodiments, the

o endonuclease is a Cas9 homolog or ortholog.

In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease is further modified to alter the activity of the protein. In some embodiments, the Cas9 endonuclease is a catalytically inactive Cas9. For example, dCas9 contains mutations of catalytically active residues (D10 and H840) and does not have nuclease activity.

5 Alternatively or in addition, the Cas9 endonuclease may be fused to another protein or portion thereof. In some embodiments, dCas9 is fused to a repressor domain, such as a KRAB domain. In some embodiments, such dCas9 fusion proteins are used with the constructs described herein for multiplexed gene repression (e.g. CRISPR interference (CRISPRi)). In some embodiments, dCas9 is fused to an activator domain, such as VP64 or o VPR. In some embodiments, such dCas9 fusion proteins are used with the constructs

described herein for gene activation (e.g., CRISPR activation (CRISPRa)). In some embodiments, dCas9 is fused to an epigenetic modulating domain, such as a histone demethylase domain or a histone acetyltransferase domain. In some embodiments, dCas9 is fused to a LSD1 or p300, or a portion thereof. In some embodiments, the dCas9 fusion is 5 used for CRISPR-based epigenetic modulation. In some embodiments, dCas9 or Cas9 is fused to a Fokl nuclease domain. In some embodiments, Cas9 or dCas9 fused to a Fokl nuclease domain is used for genome editing. In some embodiments, Cas9 or dCas9 is fused to a fluorescent protein (e.g., GFP, RFP, mCherry, etc.). In some embodiments, Cas9/dCas9 proteins fused to fluorescent proteins are used for labeling and/or visualization of genomic0 loci or identifying cells expressing the Cas endonuclease.

Provided below are amino acid sequences of two exemplary Cas9 proteins: DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKYPTIYH LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQK KAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENE DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDHIVPQSFLADDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPALESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSM PQV IVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL KSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQI SEFSKRVILADANLDKVLSA YNKHRDKP IREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRID LSQLGGD (SEQ ID NO : 4 )

MDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKRTA RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP ILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKK LKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD (SEQ ID NO : 5 ) Alternatively or in addition, the Cas endonuclease is a Cpfl nuclease. In some embodiments, the host cell expresses a Cpfl nuclease derived from Provetella spp. or Francisella spp. In some embodiments, the nucleotide sequence encoding the Cpfl nuclease may be codon optimized for expression in a host cell. Exemplary Cpfl nucleases can be found under, e.g., GenBank accession no. ASK09413 and GenBank accession no. A0Q7Q2. (C) Preparation of Fusion Polypeptides

Any of the fusion polypeptides can be prepared via routine recombinant

technology. For example, the coding sequences of the 53BP1 DN variant and the gene- editing nuclease enzyme can be fused in-frame via routine technology, either directed or via any linker, and cloned into a suitable vector and the recombinant protein generated.

Alternatively, the fusion polypeptide and gene editing nuclease can be synthesized using peptide synthesis technology. In other circumstances, the 53BP1 DN fusion with a gene editing nuclease can be expressed as mRNA. The coding sequence can also be in operable in DNA or RNA viruses, expressed linkage to a suitable promoter {e.g., a mammalian promoter) for expression of the fusion polypeptide in a suitable host cell.

Vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329: 840) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, ct al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The vectors of the present disclosure are capable of directing expression of the nucleic acid preferentially in a particular cell type {e.g., tissue- specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue {e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term "cell type specific" as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g. , immunohistochemical staining.

Non-limiting examples of viral vectors include, but are not limited to, retroviral vectors (e.g. , lentiviral vectors or gammaretroviral vectors), adenoviral vectors, adeno- associated viral vectors (AAV), and hybrid vectors (containing components from different viral genomes). Additional examples of viral vectors are provided in US Patent Patent No. 5,698,443, US Patent No. 5,650,309, and US Patent No, 5,827,703, the relevant disclosures of each of which are herein incorporated by reference for the purpose and subject matter referenced herein.

Any of the nucleic acids encoding the fusion polypeptides disclosed herein, vectors comprising such, and host cells comprising the vectors are within the scope of the present disclosure.

Two exemplary fusion polypeptides, both containing the 53BP1 DN1S variant linked to a Cas9 protein via a TGS linker, are provided below (including both amino acid sequences and nucleotide sequences):

DKKYSIGTDIGTNSVGWAVITDEYKVPSKKFKVTGNTDRHSIKKNTIGATTFDSGETAEATRTKRT ARRRYTRRKNRICYTQEIFSNEMAKVDDSFFHRTEESFTVEEDKKHERHP IFG IVDEVAYHEKYP TIYHTRKKTVDSTDKADTRTIYTATAHMIKFRGHFTIEGDTNPDNSDVDKTFIQTVQTYNQTFEEN PINASGVDAKAITSARTSKSRRTENTIAQTPGEKKNGTFGNTIATSTGTTPNFKSNFDTAEDAKTQ TSKDTYDDDTDNTTAQIGDQYADTFTAAKNTSDAITTSDITRVNTEITKAPTSASMIKRYDEHHQD TTTTKATVRQQTPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPITEKMDGTEETTVKTNRED TTRKQRTFDNGS IPHQIHTGETHAITRRQEDFYPFTKDNREKIEKITTFRIPYYVGPTARGNSRFA WMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNTPNEKVTPKHSTTYEYFTVYNETTKVKY VTEGMRKPAFTSGEQKKAIVDTTFKTNRKVTVKQTKEDYFKKIECFDSVEI SGVEDRFNASTGTYH DTTKI IKDKDFTDNEENEDITEDIVTTTTTFEDREMIEERTKTYAHTFDDKVMKQTKRRRYTGWGR TSRKTINGIRDKQSGKTITDFTKSDGFANRNFMQTIHDDSTTFKEDIQKAQVSGQGDSTHEHIANT AGSPAIKKGITQTVKVVDETVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKETGS QITKEHPVENTQTQNEKTYTYYTQNGRDMYVDQETDINRTSDYDVDHIVPQSFTADDS IDNKVTTR SDKNRGKSDNVPSEEVVKKMKNYWRQTTNAKTITQRKFDNTTKAERGGTSETDKAGFIKRQTVETR QITKHVAQITDSRMNTKYDENDKTIREVKVITTKSKTVSDFRKDFQFYKVREINNYHHAHDAYTNA WGTATIKKYPATESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITTANGEI RKAPTIETNGETGEIVWDKGRDFATVRKVTSMPQVNIVKKTEVQTGGFSKESITPKRNSDKTIARK KDWDPKKYGGFDSPTVAYSVTVVAKVEKGKSKKTKSVKETTGITIMERSSFEKNPIDFTEAKGYKE VKKDTI IKTPKYSTFETENGRKRMTASAGETQKGNETATPSKYVNFTYTASHYEKTKGSPEDNEQK QTFVEQHKHYTDEI IEQI SEFSKRVITADANTDKVTSAYNKHRDKP IREQAENI IHTFTTTNTGAP AAFKYFDTTIDRKRYTSTKEVTDATTIHQS ITGTYETRIDTSQTGGD TGSTGSTGSTGSMGPHGHV LHRHMRTIREVRTTVTRVITDVYYVDGTEVERKVTEETEEPIVECQECETEVSPSQTGGSSGDTGD ISSFSSKASSLHRTSSGTSTSAMHSSGSSGKGAGPTRGKTSGTEPADFALPSSRGGPGKTSPRKGV SQTGTPVCEEDGDAGTGIRQGGKAPVTPRGRGRRGRPPSRTTGTRETAVPGPLGMEDI SPNLSPDD KSFSRVVPRVPDSTRRTDVGAGALRRSDSPEIPFQAAAGPSDGLDASSPGNSFVGTRVVAKWSSNG YFYSGKITRDVGAGKYKLTFDDGYECDVLGKDITLCDP IPTDTEVTATSEDEYFSAGVVKGHRKES GETYYS IEKEGQRKWYKRMAVITSLEQGNRTREQYGTGPYEAVTPTTKAADISLDNLVEGKRKRRS NVSSPATPTASSS (SEQ ID NO : 6 )

GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAG GTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTG CTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAG AACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTG GAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTG GCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTG CGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCC GACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATC AACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATC GCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAAC TTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGAC AACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTG CTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGAC GAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTC TTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATC AAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAG CAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTAC GTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGG AACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAA GTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTG CTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGAC TCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTT GAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTG AAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGC AAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGC CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAAT CTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATG GGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAAC AGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTG GAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAG GAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGGCGGACGACTCC ATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTG AAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACC AAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAG ATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGG GAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGC GAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTAC CCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG CAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACC

CTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAG GGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAG ACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGG GACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAG GGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAG

AATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTG GCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGAT AATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTC TCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCC ATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTAC TTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGC ATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACACAGGGrCCACAGGATCCACAGGC AGCACAGGGAGCATGGGACCACATGGCCATGTCTTACATCGTCACATGAGAACAATCCGGGAAGTACGCACACTT GTCACTCGTGTCATTACAGATGTGTATTATGTGGATGGAACAGAAGTAGAAAGAAAAGTAACTGAGGAGACTGAA GAGCCAATTGTAGAGTGTCAGGAGTGTGAAACTGAAGTTTCCCCTTCACAGACTGGGGGCTCCTCAGGTGACCTG GGGGATATCAGCTCCTTCTCCTCCAAGGCATCCAGCTTACACCGCACATCAAGTGGGACAAGTCTCTCAGCTATG CACAGCAGTGGAAGCTCAGGGAAAGGAGCCGGACCACTCAGAGGGAAAACCAGCGGGACAGAACCCGCAGATTTT GCCTTACCCAGCTCCCGAGGAGGCCCAGGAAAACTGAGTCCTAGAAAAGGGGTCAGTCAGACAGGGACGCCAGTG TGTGAGGAGGATGGTGATGCAGGCCTTGGCATCAGACAGGGAGGGAAGGCTCCAGTCACGCCTCGTGGGCGTGGG CGAAGGGGCCGCCCACCTTCTCGGACCACTGGAACCAGAGAAACAGCTGTGCCTGGCCCCTTGGGCATAGAGGAC ATTTCACCTAACTTGTCACCAGATGATAAATCCTTCAGCCGTGTCGTGCCCCGAGTGCCAGACTCCACCAGACGA ACAGATGTGGGTGCTGGTGCTTTGCGTCGTAGTGACTCTCCAGAAATTCCTTTCCAGGCTGCTGCTGGCCCTTCT GATGGCTTAGATGCCTCCTCTCCAGGAAATAGCTTTGTAGGGCTCCGTGTTGTAGCCAAGTGGTCATCCAATGGC TACTTTTACTCTGGGAAAATCACACGAGATGTCGGAGCTGGGAAGTATAAATTGCTCTTTGATGATGGGTACGAA TGTGATGTGTTGGGCAAAGACATTCTGTTATGTGACCCCATCCCGCTGGACACTGAAGTGACGGCCCTCTCGGAG GATGAGTATTTCAGTGCAGGAGTGGTGAAAGGACATAGGAAGGAGTCTGGGGAACTGTACTACAGCATTGAAAAA GAAGGCCAAAGAAAGTGGTATAAGCGAATGGCTGTCATCCTGTCCTTGGAGCAAGGAAACAGACTGAGAGAGCAG TATGGGCTTGGCCCCTATGAAGCAGTAACACCTCTTACAAAGGCAGCAGATATCAGCTTAGACAATTTGGTGGAA GGGAAGCGGAAACGGAGATCTAACGTCAGCTCCCCAGCCACCCCTACTGCCTCCTCGAGC (SEQ ID NO : 7 )

MDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKR TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP IFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NP INASGVDAKAILSARLSKSRRLENLI AQLPGEKKNGLFGNLI ALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP ILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVK YVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI AN LAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLT RSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYS IMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP QVNIVKKTEVQTGGFSKESILPKRNSDKLI AR KKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGI TIMERS SFEKNP IDFLEAKGYK EVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEI IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE I IHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDTGS TGSTGSTGSMGPHGH VLHRHMRTIREVRTLVTRVITDVYYVDGTEVERKVTEETEEP IVECQECETEVSPSQTGGSSGDLG DISSFSSKASSLHRTSSGTSLSAMHSSGSSGKGAGPLRGKTSGTEPADFALPSSRGGPGKLSPRKG VSQTGTPVCEEDGDAGLGIRQGGKAPVTPRGRGRRGRPPSRTTGTRETAVPGPLGIEDISPNLSPD DKSFSRWPRVPDSTRRTDVGAGALRRSDSPEIPFQAAAGPSDGLDASSPGNSFVGLRWAKWSSN GYFYSGKITRDVGAGKYKLLFDDGYECDVLGKDILLCDPIPLDTEVTALSEDEYFSAGWKGHRKE SGELYYSIEKEGQRKWYKRMAVILSLEQGNRLREQYGLGPYEAVTPLTKAADISLDNLVEGKRKRR SNVSSPATPTASSS (SEQ ID NO : 8 )

ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATAT AAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT CTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGG AAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA

CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAA GTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGAT TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAAT CCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCT ATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTC

ATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCT AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTA GATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT TTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTAC GATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTT ATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGA CAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCA TGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACA AAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTT GATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATT ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTA TTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCT GGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT AGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCA AATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTA ATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAA AATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGAC CAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGAT TCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTA GTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTA ACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGC CAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATT CGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTA CGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAA TATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCT GAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATT ACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGAT AAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTA CAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGAC TGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAA AAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAA AAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAG CTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAA GATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAA TTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAA CCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAA TATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAA TCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACACGGGrrCAaCrGGArCTACG GGAaGTACCGGCAGCArGGGGCCTCATGGCCACGTTCTGCACCGTCACATGCGCACGATTCGTGAAGTACGCACA CTGGTCACCCGCGTTATTACAGACGTGTACTATGTGGATGGAACAGAGGTAGAGCGTAAAGTCACCGAGGAAACG GAAGAGCCTATCGTTGAATGTCAGGAATGTGAAACTGAAGTATCCCCCAGTCAGACCGGAGGGTCTTCAGGTGAT CTTGGGGATATCTCGAGCTTCTCTTCCAAAGCAAGTTCGTTGCATCGCACAAGCTCTGGAACGTCCTTGAGTGCA ATGCACAGTTCTGGTAGTTCGGGGAAAGGAGCCGGACCACTGCGTGGTAAGACCTCTGGCACCGAACCTGCAGAT TTTGCATTGCCATCCAGCCGCGGAGGGCCTGGCAAACTGTCACCACGTAAGGGGGTGAGCCAAACCGGCACACCT GTTTGTGAGGAAGATGGAGATGCTGGTTTAGGCATCCGCCAAGGTGGCAAGGCACCAGTCACCCCCCGTGGCCGT

GGACGCCGCGGACGCCCCCCATCTCGCACGACGGGTACGCGTGAGACTGCCGTCCCGGGACCCTTGGGCATCGAG GATATTTCACCGAACTTGAGCCCAGACGACAAAAGCTTTAGCCGCGTGGTACCCCGCGTGCCCGATAGCACACGC CGTACGGACGTCGGAGCTGGTGCATTGCGCCGCTCGGACAGCCCCGAAATCCCATTCCAAGCAGCCGCCGGACCT TCGGACGGCTTGGATGCATCGTCACCGGGGAACAGTTTTGTAGGGCTTCGCGTCGTAGCTAAGTGGTCTTCGAAT GGCTACTTTTATAGTGGTAAGATCACCCGTGATGTTGGCGCAGGTAAATACAAACTGTTGTTCGACGACGGATAC

GAATGTGATGTCTTAGGGAAGGACATCCTTCTTTGTGATCCAATTCCTTTGGACACGGAGGTCACCGCTTTGTCC GAAGACGAGTACTTTAGTGCGGGAGTCGTTAAGGGTCATCGTAAGGAAAGTGGAGAGTTGTATTACTCCATCGAA AAAGAGGGTCAACGCAAGTGGTACAAGCGCATGGCCGTAATTCTGTCCCTGGAACAGGGGAACCGCCTGCGTGAG CAGTACGGCCTGGGACCTTACGAGGCAGTGACACCACTTACGAAAGCAGCCGATATCTCATTGGACAACTTGGTC GAAGGTAAGCGTAAACGTCGTTCAAACGTGTCGTCCCCCGCGACACCTACTGCCTCATCATCG (SEQ ID NO: 9)

The sequences above in boldface and italicized refer to the linker amino acid and coding nucleotide sequences. The option at the N-terminal or 5' end of the linker sequences are the Cas9 protein and the option at the C-terminal or 3' end of the linker sequences are the 53BP1 DN1 variant. Uses of Fusion Polypeptides in Gene Editing

Any of the fusion polypeptides disclosed herein can be used in gene editing, following routine methodology associated with the specific gene-editing nuclease contained in the fusion polypeptide. For example, the fusion polypeptide, or a suitable vector encoding such, can be delivered into host cells where gene-editing is needed.

gRNAs specific to the genetic site to be edited and optionally a template nucleic acid guiding homologous recombination can be co-delivered into the host cells via routine methods, e.g., electroporation of nucleic acid or RNP complex, or viral particle infection.

In one example, the fusion polypeptide comprises a Cas protein (a Cas9 protein or a homolog thereof such as Casl2) fused to a 53BP1 DN variant. Such a fusion

polypeptide can be used in the CRISPR-Cas system to edit a specific gene of interest.

CRISPR-Cas system has been successfully utilized to edit the genomes of various organisms, including, but not limited to bacteria, humans, fruit flies, zebra fish and plants. See, e.g., Jiang et al., Nature Biotechnology (2013) 31(3):233; Qi et al, Cell (2013)

5: 1173; DiCarlo et al., Nucleic Acids Res. (2013) 7:4336; Hwang et al., Nat. Biotechnol (2013), 3:227); Gratz et al., Genetics (2013) 194: 1029; Cong et al., Science (2013)

6121:819; Mali et al., Science (2013) 6121:823; Cho et al. Nat. Biotechnol (2013) 3: 230; and Jiang et al., Nucleic Acids Research (2013) 41(20):el88.

The method disclosed herein may utilize the CRISPR/Cas9 system that hybridizes with a target sequence in a gene of interest, where the CRISPR/Cas9 system comprises a Cas9/53BP1 DN variant fusion polypeptide and an engineered crRNA/tracrRNA (or a single guide RNA). CRISPR/Cas9 complex can bind to the genetic site to be edited and allow the cleavage of the target site, thereby modifying the gene of interest.

The CRISPR/Cas system of the present disclosure may bind to and/or cleave the gene of interest in a coding or non-coding region, within or adjacent to the gene, such as, for example, a leader sequence, trailer sequence or intron, or within a non-transcribed region, either upstream or downstream of the coding region. The guide RNAs (gRNAs) used in the present disclosure may be designed such that the gRNA directs binding of the Cas9-gRNA complexes to a pre-determined cleavage sites (target site) in a genome. The cleavage sites may be chosen so as to release a fragment that contains a region of unknown sequence, or a region containing a SNP, nucleotide insertion, nucleotide deletion, rearrangement, etc. Cleavage of a gene region may comprise cleaving one or two strands at the location of the target sequence by the Cas enzyme. In one embodiment, such, cleavage can result in decreased transcription of a target gene. In another embodiment, the cleavage can further comprise repairing the cleaved target polynucleotide by homologous recombination with an 5 exogenous template polynucleotide, wherein the repair results in an insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. It is expected that the repair efficiency via homologous recombination would be enhanced when a Cas/53BP1 DN variant fusion polypeptide is used.

The terms "gRNA" and "guide RNA" may be used interchangeably throughout and o refer to a nucleic acid comprising a sequence that determines the specificity of a Cas DNA binding protein of a CRISPR/Cas system. A gRNA hybridizes to (complementary to, partially or completely) a target nucleic acid sequence in the genome of a host cell. The gRNA or portion thereof that hybridizes to the target nucleic acid may be between 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length. In some embodiments, the5 gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is between 10-30, or between 15-25, nucleotides in length.

In addition to a sequence that binds to a target nucleic acid, in some embodiments, the gRNA also comprises a scaffold sequence. Expression of a gRNA encoding both a sequence o complementary to a target nucleic acid and scaffold sequence has the dual function of both binding (hybridizing) to the target nucleic acid and recruiting the endonuclease to the target nucleic acid, which may result in site-specific CRISPR activity. In some embodiments, such a chimeric gRNA may be referred to as a single guide RNA (sgRNA).

As used herein, a "scaffold sequence," also referred to as a tracrRNA, refers to a

5 nucleic acid sequence that recruits a Cas endonuclease to a target nucleic acid bound

(hybridized) to a complementary gRNA sequence. Any scaffold sequence that comprises at least one stem loop structure and recruits an endonuclease may be used in the genetic elements and vectors described herein. Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinek, et al. Science (2012)

0 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Application No.

WO2014/093694, and PCT Application No. WO2013/176772. In some embodiments, the gRNA sequence does not comprises a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence and recruit 5 the endonuclease to the target nucleic acid.

In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to a target nucleic acid (see also US Patent 8,697,359, which is incorporated by reference for its teaching of complementarity of a gRNA sequence with a target polynucleotide sequence). It o has been demonstrated that mismatches between a CRISPR guide sequence and the target nucleic acid near the 3' end of the target nucleic acid may abolish nuclease cleavage activity (Upadhyay, et al. Genes Genome Genetics (2013) 3(12):2233-2238). In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3' end of the target nucleic5 acid {e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3' end of the target nucleic acid).

The target nucleic acid is flanked on the 3' side by a protospacer adjacent motif (PAM) that may interact with the endonuclease and be further involved in targeting the endonuclease activity to the target nucleic acid. It is generally thought that the PAM sequence flanking the target nucleic acid depends on the endonuclease and the source from o which the endonuclease is derived. For example, for Cas9 endonucleases that are derived from Streptococcus pyogenes, the PAM sequence is NGG. For Cas9 endonucleases derived from Staphylococcus aureus, the PAM sequence is NNGRRT. For Cas9 endonucleases that are derived from Neisseria meningitidis, the PAM sequence is NNNNGATT. For Cas9 endonucleases derived from Streptococcus thermophilus, the PAM sequence is NNAGAA. 5 For Cas9 endonuclease derived from Treponema denticola, the PAM sequence is NAAAAC.

For a Cpf 1 nuclease, the PAM sequence is TTN.

In some embodiments, genetically engineering a cell also comprises introducing a Cas endonuclease into the cell. In some embodiments, the Cas endonuclease and the nucleic acid encoding the gRNA are provided on the same nucleic acid (e.g., a vector). In some

0 embodiments, the Cas endonuclease and the nucleic acid encoding the gRNA are provided on different nucleic acids (e.g., different vectors). Alternatively or in addition, the Cas endonuclease may be provided or introduced into the cell in protein form. The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode one or more components of a CRISPR/Cas9 complex, wherein the vector comprises a polynucleotide encoding (i) a (CRISPR)-Cas system guide RNA that hybridizes to the gene of interest and (ii) a Cas9/53BP1 DN variant fusion

5 polypeptide.

Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding CRISPR/Cas9 in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR-Cas system to cells in culture, or in a host organism. Non-viral vector delivery systems include o DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures.

Viral vectors can be administered directly to patients (in vivo) or they can be used to5 manipulate cells in vitro or ex vivo, where the modified cells may be administered to patients.

In one embodiment, the present disclosure utilizes viral based systems including, but not limited to retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Furthermore, the present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. Preferably, the vector used for the o expression of a CRISPR-Cas system of the present disclosure is a lentiviral vector.

In one embodiment, the disclosure provides for introducing one or more vectors encoding CRISPR-Cas into eukaryotic cell. The cell can be a cancer cell. Alternatively, the cell is a hematopoietic cell, such as a hematopoietic stem cell. Examples of stem cells include pluripotent, multipotent and unipotent stem cells. Examples of pluripotent stem cells 5 include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells and induced pluripotent stem cells (iPSCs). In a preferred embodiment, the disclosure provides introducing CRISPR-Cas9 into a hematopoietic stem cell.

The vectors of the present disclosure are delivered to the eukaryotic cell in a subject. Modification of the eukaryotic cells via CRISPR/Cas9 system can takes place in a cell

0 culture, where the method comprises isolating the eukaryotic cell from a subject prior to the modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to the subject. General techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press;

Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.

1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and

C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Example 1: Identification of HDR- Enhancer Fragment of 53BP1

Several dominant negative (DN) p53 binding protein 1 (53BP1) mutants were designed based on presence of binding to DNA double strand breaks (DSB), but inability to recruit other non-homologous end joining (NHEJ). Five DN 53BP1 mutants were designed as shown in Figure 1, panel A. Nucleic acids encoding these DN mutants (with an HA tag) were cloned into a lentiviral vector, which was delivered into host cells (e.g. , HeLa cells) for expression. Expression of the HA-tagged DN mutants were examined by Western blot and the results are shown in Figure 1, panels B and C. The results show that the vectors carrying the DN1, DNls, DN3, and DN4 mutants expressed the recombinant 53BP1 mutants at the correct size. Figure 1, panel B. Expression of DN2 was observed at higher exposure. Figure 1, panel C. Expression of the 53BP1 DN mutants were also observed by in

immunofluorescence (IF) imaging via detection of HA and DAPl . Representative immunofluorescence (IF) images showed HA tagged DN1S and endogenous 53BP1 recruitment to the irradiation-induced DNA break site. Mock has no HA whereas HA co- localizes with or even displaces all the endogenous 53BP1 foci at low or high expression of DN1S, respectively. Lesser number of endogenous 53BP1 was observed in cells expressing DN1 mutant, indicating that the mutant successfully replaced endogenous 53BP1 in DNA damage foci.

Next, recruitment of the 53BP1 DN mutants to DNA damage foci in HeLa cells was examined. HeLa cells expression the 53BP1 DN mutants noted above were stained for DAPl (damage response protein 1), endogenous 53BP1, and HA tag (indicating 53BP1 DN mutants) and analysed by immunofluorescence (IF). Representative immunofluorescence (IF) images showing HA tagged DN1/DN1S and endogenous 53BP1 recruitment to the irradiation-induced DNA break site. Mock has no HA whereas HA co-localizes with all the endogenous 53BP1 foci in DN1/DN1S arm. See also Figure 1, panel D. Similarly, representative immunofluorescence (IF) images showing HA tagged DN1/DN1S and RIF- 1 or γΗ2ΑΧ recruitment to the irradiation-induced DNA break site. Mock has no HA and many RIF- 1 foci in each cell whereas DN1/DN1S arm with HA+ cells has reduced/no RIF-1 or γΗ2ΑΧ foci in each cell. See also Figure 1, panel E and panel G. BRCA1 is a key protein in the homology directed repair. Similar results were observed in connection with BRCA-1 recruitment. It is normally present in S phage foci in untreated cells. Representative immunofluorescence (IF) images showing HA tagged DN1S and BRCA-1 recruitment to the DNA break site. Mock has no HA and few BRCA-1 foci in each cell whereas DN1S arm with HA+ cells has higher BRCA-1 foci in each cell. The percentage of cells with BRCA1 foci was significantly increased in HeLa cells containing 53BP1 DN1, DNls, DN2, and DN4 mutants with DN1 and DNls having the highest number of cells with BRCA1. Figure 1, panel F.

The results from this study indicate that the designed 53BP1 DN mutants expressed in host cells and are recruited to DNA damage foci in the host cells expressing such. The results also show that the 53BP1 DN mutants can replace endogenous 53BP1 proteins at the DNA damage sites, indicating that the mutants can block activity of the endogenous 53BP1.

Example 2: Cas9-53BP1 DN Fusion Proteins Inhibited NHEJ and Enhanced

Homologous Recombination

Fusion proteins containing a 53BP1 DN mutant and Cas9 were constructed via routine recombinant technology. Figure 2, panel A. Plasmids were generated to express each of the four Cas9-53BP1 DN fusion proteins and transfected into 293T cells carrying the Traffic

Light Reporter. Expression of the fusion proteins were detected by Western blot analysis as shown in Figure 2, panel B. Representative immunofluorescence (IF) imaging shows HA tagged DN1S or dCas9-DNlS/gRNA and endogenous 53BP1 recruitment to the DNA break site. Figure 2, panel E. Panels C and D show the level of cells having >1 HA foci and > 53BP1 foci, respectively. It was also observed that the 53BP1 DN mutants, either alone or in fusion with Cas9, locally inhibited NHEJ and reduced cellular toxicity. Figure 2 panels F and G. Ionizing radiation (IR) induces DSBs and if cellular NHEJ -based repair pathways are compromised, cell undergo apoptosis. Cas9-DN 53BP1 fusion only inhibits NHEJ at Cas9 cut sites and does not sensitize cells to apoptosis in Figure 2, panels F-G.

The Traffic Light Reporter (TLR) system in host cells such as 293T cells was used to examine the effect of Cas9-53BP1 DN fusion proteins in NHEJ repair and HDR. As illustrated in Figure 3, panel A, a TLR reporter system was introduced into 293T cells. The TLR system includes a venus reporter (green) for detecting homologous recombination and a red fluorescent protein (RFP) for detecting NHEJ. Upon introducing a Cas9/53BP1 DN fusion protein together with the gRNA shown in Figure 3, panel A, the Cas9 enzyme creates a DSB at the site directed by the gRNA. If repair of the DSB is via NHEJ, expression of the the otherwise Out of frame' RFP reporter via NHEJ would a third of the time cause a frame shift, leading to expression of the RFP reporter in frame, and making cells fluoresce red. If repair of the DSB is via HDR, the venus reporter targeted would become functional and the RFP reporter would be rendered nonfunctional during HDR, resulting in expression of only the venus reporter, making cells with HDR fluoresce green. Figure 3, panel B.

Results from this study show that, relative to the Cas9 protein, the gene editing efficiency via NHEJ was reduced and the gene editing efficiency via HDR was enhanced, particularly when the Cas9-DN1 or Cas9-DN1S fusion proteins were used. Furthermore, use of different tags or linkers, or fusing the DN at the amino terminus or carboxy terminus of Cas9 all showed increased HDR, Figure 3D. These results were observed in various cell lines and at different target genes using different Cas9 nucleases. At rare loci, where HDR is not improved, NHEJ is still very highly significantly reduced, and this is clinically relevant to not cause inadvertent indels or mutations. Figure 4, panels A-C.

Similar results were observed in patient derived EBV transformed B lymphocyte cells using AAVS 1 gRNA and Cas9-DN1S fusion protein to target the normal CD 18 gene into the AAVS 1 locus. CD 18 deficiency leads to leukocyte adhesion defect and therefore inability of leukocytes to adhere and kill invading organisms, resulting in immune deficiency. This disease was chosen for two reasons: CD18 surface expression can be detected by flow cytometry, making the readout possible at a single cell level. Second, CD 18 deficiency correction requires high level CD 18 expression (low levels of CD 18 expression from lentivirus vectors do not correct the defect in dogs with LAD, while high expression corrects the disease). It was observed that Cas9-DN resulted in higher HDR and higher bi-allelic HDR, resulting is very high CD 18 expression, Figure 5 A-C.

The cumulative data from Figure 5 shows that when the DN1S is fused to different site specific nucleases (either SpCas9 or SaCas9), there is a highly significant reduction in site- specific NHEJ. This results in a highly significant increase in HDR. Even occasional loci that show no increase in HDR show a highly significant reduction in NHEJ (error-creating) repair. Moreover, there is not only a quantitative, but also a qualitative improvement in HDR, as observed with higher biallelic HDR in patient derived B lymphocytes. Furthermore, while we see improved HDR and reduced NHEJ at Cas9 on-target sites, we observe less off target cutting of Cas9-DN at known off target sites described by this gRNA/Cas9 at this locus, Figure 6, suggesting that the fusion of DN 53BP1 to a gene editing nuclease reduces its geno toxicity and lowers its affinity to off target sites.

5 Overall, we observe that DN 53BP1 fusion to Cas9 gene editing nucleases a) inhibits

NHEJ only at sites where gene editing nucleases are designed to cut, b) increases both the quantity of HDR (% cells with HDR) and the quality of HDR (bi-allelic HDR), and c) reduces the off-target cutting of Cas9.

OTHER EMBODIMENTS

o All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

5 From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

o While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those 5 skilled in the art will readily appreciate that all parameters, dimensions, materials, and

configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many

0 equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

CLAIMS What Is Claimed Is:

1. A fusion polypeptide, comprising a gene-editing nuclease enzyme and a dominant-negative variant of a p53 binding protein 1 (53BP1), wherein the dominant- negative variant of 53BP1 is a truncated 53BP1, which comprises (a) deletion in a docking domain, (b) a deletion of a BRCT domain, or (c) both (a) and (b).

2. The fusion polypeptide of claim 1, wherein the dominant negative variant of 53BP1 comprises (a) a deletion of region 1-1231 of SEQ ID NO: l or a portion thereof, (b) a deletion of region 1711-1972 of SEQ ID NO: l or a portion thereof, or (c) both (a) and (b).

3. The fusion polypeptide of claim 1 or claim 2, wherein the gene-editing nuclease enzyme is a Cas9 enzyme, a Casl2 enzyme, a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN) or a meganuclease.

4. The fusion polypeptide of claim 3, wherein the Cas9 enzyme comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

5. The fusion polypeptide of any one of claims 1-4, wherein the dominant- negative variant of 53BP1 comprises region 1480-1644 of SEQ ID NO: l.

6. The fusion polypeptide of claim 5, wherein the dominant-negative variant of 53BP1 comprises region 1231-1644 of SEQ ID NO: l, region 1231-1711 of SEQ ID NO: l, or 1480-1711 of SEQ ID NO: 1.

7. The fusion polypeptide of claim 6, wherein the dominant- negative variant of 53BP1 consists of 1231-1711 of SEQ ID NO: 1, 1231-1644 of SEQ ID NO: 1, or 1480-1711 of SEQ ID NO: 1.

8. The fusion polypeptide of any one of claims 1-7, wherein the gene-editing nuclease enzyme is covalently linked directly to the dominant-negative variant of 53BP1.

9. The fusion polypeptide of any one of claims 1-8, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8.

10. A nucleic acid, comprising a nucleotide sequence coding for a fusion polypeptide or mRNA of any one of claims 1-9.

11. A vector comprising the nucleic acid of claim 10, wherein the nucleotide sequence coding for the fusion polypeptide is in operable linkage to a promoter.

12. The vector of claim 11, wherein the promoter is a mammalian promoter.

13. The vector of claim 11 or claim 12, which is a viral vector.

14. The vector of claim 13, wherein the viral vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a hybrid vector.

15. The vector of claim 14, wherein the viral vector is a retroviral vector, which is a lentiviral vector, a foamy virus vector or a gamma retrovirus vector.

16. The vector of any one of claims 11-15, further comprising a nucleotide sequence coding for a guide RNA.

17. A method for enhancing homology directed DNA repair (HDR) in gene editing of a cell, comprising introducing into a cell (a) the fusion polypeptide of any one of claims 1-9 or a vector that comprises a nucleotide sequence coding for the fusion

polypeptide.

18. The method of claim 17, further comprising introducing into the cell (b) a guide RNA targeting a gene of interest.

19. The method of claim 18, wherein the guide RNA is a single guide RNA.

20. The method of any one of claims 17-19, wherein the cell is a mammalian cell.

21. The method of claim 20, wherein the mammalian cell is a human cell.

22. The method of any one of claims 17-21, wherein (a) and (b) are introduced into the cell by delivering a vector that expresses both the fusion polypeptide and the guide RNA into the cell.

23. The method of any one of claims 17-22, wherein the method further comprising introducing into the cell a template nucleic acid, which comprises homologous arms flanking a cleavage site in the gene of interest directed by the guide RNA.