Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR)1,2,3. However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ)4. We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells4,5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.
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Doudna, J.A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Cox, D.B., Platt, R.J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Chandrasegaran, S. & Carroll, D. Origins of Programmable Nucleases for Genome Engineering. J. Mol. Biol. 428 5 Pt B, 963–989 (2016).
Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2016).
Panier, S. & Boulton, S.J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).
Ernst, A. et al. A strategy for modulation of enzymes in the ubiquitin system. Science 339, 590–595 (2013).
Escribano-Díaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883 (2013).
Feng, L., Fong, K.W., Wang, J., Wang, W. & Chen, J. RIF1 counteracts BRCA1-mediated end resection during DNA repair. J. Biol. Chem. 288, 11135–11143 (2013).
Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).
Botuyan, M.V. et al. Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).
Dikic, I., Wakatsuki, S. & Walters, K.J. Ubiquitin-binding domains - from structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 (2009).
Orthwein, A. et al. A mechanism for the suppression of homologous recombination in G1 cells. Nature 528, 422–426 (2015).
O'Donnell, L. et al. The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination. Mol. Cell 40, 619–631 (2010).
Xie, A. et al. Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair. Mol. Cell 28, 1045–1057 (2007).
Moynahan, M.E., Chiu, J.W., Koller, B.H. & Jasin, M. Brca1 controls homology-directed DNA repair. Mol. Cell 4, 511–518 (1999).
Srivastava, M. et al. An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Cell 151, 1474–1487 (2012).
Chu, V.T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).
Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).
Pinder, J., Salsman, J. & Dellaire, G. Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic Acids Res. 43, 9379–9392 (2015).
Agudelo, D. et al. Marker-free coselection for CRISPR-driven genome editing in human cells. Nat. Methods 14, 615–620 (2017).
Maggio, I. & Gonçalves, M.A. Genome editing at the crossroads of delivery, specificity, and fidelity. Trends Biotechnol. 33, 280–291 (2015).
Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015).
Wang, J. et al. Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Res. 22, 1316–1326 (2012).
Verma, P. & Greenberg, R.A. Noncanonical views of homology-directed DNA repair. Genes Dev. 30, 1138–1154 (2016).
Davis, L. & Maizels, N. Two distinct pathways support gene correction by single-stranded donors at DNA nicks. Cell Rep. 17, 1872–1881 (2016).
Richardson, C.D., Ray, G.J., DeWitt, M.A., Curie, G.L. & Corn, J.E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).
Sfeir, A. & Symington, L.S. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway? Trends Biochem. Sci. 40, 701–714 (2015).
Muñoz, M.C. et al. RING finger nuclear factor RNF168 is important for defects in homologous recombination caused by loss of the breast cancer susceptibility factor BRCA1. J. Biol. Chem. 287, 40618–40628 (2012).
Xiong, X. et al. 53BP1 promotes microhomology-mediated end-joining in G1-phase cells. Nucleic Acids Res. 43, 1659–1670 (2015).
Gunn, A. & Stark, J.M. I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol. Biol. 920, 379–391 (2012).
Bennardo, N., Cheng, A., Huang, N. & Stark, J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).
Richardson, C.D. et al. CRISPR-Cas9 genome editing in human cells works via the Fanconi anemia pathway. Preprint at bioRxiv https://www.biorxiv.org/content/early/2017/05/09/136028 (2017).
Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).
Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Tonikian, R., Zhang, Y., Boone, C. & Sidhu, S.S. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat. Protoc. 2, 1368–1386 (2007).
Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).
We are grateful to R. Szilard for critical reading of the manuscript. We thank J. Stark (City of Hope) for the DR- and EJ2-GFP U2OS cell lines, G. Dellaire (Dalhousie University) for the LMNA assay plasmids and Y. Doyon (Université Laval) for the HIST1H2BK-mAG targeting vector. The Lenti-Cas9-2A-Blast construct was a gift from J. Moffat (University of Toronto). Clone 12CA5 was gift from M. Tyers, University of Montréal). Goat anti-GFP was a gift from L. Pelletier, Lunenfeld-Tanenbaum Research Institute. A.F.-T. was a CIHR post-doctoral fellow and A.O. was a recipient of the Terry Fox Foundation Strategic Initiative for Excellence in Radiation Research for the 21st Century at CIHR fellowship. P.A.M.-G. received a Breast Cancer postdoctoral fellowship award from the US Department of Defense (BC134020). M.D.W. held a long-term fellowship from the Human Frontier Science Program. S.M.N. receives a postdoctoral fellowship from the Dutch Cancer Society (KWF). D.D. is the Thomas Kierans Chair in Mechanisms of Cancer Development and a Canada Research Chair (Tier 1) in the Molecular Mechanisms of Genome Integrity. Work was supported by NIH grant U19 HL129902 to PMC, CIHR grants MOP111149 and MOP136956 (to S.S.S.), FDN143277 (to F.S.) and FDN143343 (to D.D.) anda Grant-in-Aid from the Krembil Foundation (to D.D.).
The use of 53BP1-blocking Ubvs for stimulating genome editing are the subject of a patent application and a license agreement.
Integrated supplementary information
Supplementary Figure 1 Additional details relevant to interpretation of the 53BP1-UbvG08 structure (related to Fig. 2)
a, Structural overlay of the newly determined 53BP1 Tudor domain, the apo 53BP1 Tudor domain (PDB 1XNI) and 53BP1 Tudor domain bound to a H4K20me2 peptide ligand (PDB 2IG0). b, Structural overlay of UbvG08 and native ubiquitin. c, Structure-guided sequence alignment of UbvG08 and native ubiquitin. Secondary structural elements of native ubiquitin and UbvG08 are highlighted above and below the sequence alignment, respectively. Residue differences between ubiquitin and UbvG08 are highlighted in orange. A register shift in strand β5 is responsible for an increase in the size of the preceding loop (boxed in black) in UbvG08. d, Structural overlay of UbvG08 and native ubiquitin highlighting the difference in position of Q62 in ubiquitin with that of L62 in UbvG08 that may contribute to the differential conformation of a tight loop preceding strand β5. e, Zoom-in of the loop preceding β5 and strand β5 in ubiquitin superimposed on UbvG08. UbvG08 residues D64 (corresponding to residue E64 in ubiquitin) and K66 (corresponding to residue T66 in ubiquitin) are drastically displaced by the register shift in strand β5.
Supplementary Figure 2 Structural model validation (related to Fig. 2)
a, MBP pulldown assay where the indicated MBP-53BP1 Tudor proteins were incubated with GST-UbvG08. b, Peptide pulldown assays where the immobilized biotin-H4K20me2 peptide was used to retrieve MBP-53BP1 Tudor or the proteins as indicated. IB, immunoblot. Please note that the blots shown are cropped and the uncropped versions can be found as Supplementary Information.
Supplementary Figure 3 Effect of i53 on BRCA1 (related to Fig. 3)
Supplementary Figure 4 Control immunoblots and assessment of AAV-i53 vectors (related to Fig. 4)
a,Immunoblots of whole cell lysates prepared from U2OS DR-GFP cells transfected with the indicated siRNAs along with an I-SceI expression vector and probed with the indicated antibodies. This blot relates to the experiment shown in Fig. 4b. b, Immunoblots of whole cell lysates prepared from U2OS DR-GFP cells transfected with vectors expressing Flag-tagged i53, its DM mutant or an empty vector along with an I-SceI expression vector and probed with the indicated antibodies. This blot relates to the experiment shown in Fig. 4c. c, Control immunoblot of the experiment shown in Fig. 4d. d,e U2OS DR-GFP cells transduced with AAV particles coding for Flag-i53 (i53) or Flag-i53-DM (i53-DM) and then subjected to the DR-GFP assay following transfection of the I-SceI expression vector. In (d) the percentage of GFP-positive cells was determined 48 h post- transfection for each condition. Each pair of points are matched biological replicates; N=3. Control immunoblots showing i53 and i53-DM expression are shown in (e). Please note that the blots shown are cropped and the uncropped versions can be found as Supplementary Information.
Supplementary Figure 5 Gene targeting at the HIST1HB2K locus (related to Fig. 4).
a, b Gene targeting at the HIST1HB2K locus in 293T (a) and K562 cells (b) previously transduced with AAV coding for i53 or i53-DM (DM). The HIST1HB2K-mAG donor was introduced by nucleofection at the same time as a Cas9 RNP targeting HIST1HB2K. Control reactions where the RNP was omitted were also carried out. 72 h post-transfection, cells were analysed for mAG fluorescence by flow cytometry. Individual experiments are presented along with the mean +/− s.d., (N=3). c, Representative scatter plots for one of the biological replicates of the experiment shown in Fig. 4b.
Supplementary Figure 6 Gene targeting at the mouse Hsp90a1 locus (related to Fig. 4).
Representative scatter plots for one of the biological replicates of the experiment shown in Fig. 4c.
Supplementary Figure 7 Analysis of HDR by dsDNA and ssODNs along with primary data (related to Fig. 4).
a, K562 cells were nucleofected with ZFN mRNA (control) plus three different concentrations of i53-FLAG mRNA or DM-FLAG mRNA. Indels were then analyzed in each sample. b, BFP-to-GFP conversion by ssODN-mediated HDR in 293T (left) or MCF10a cells previously transduced with AAV coding for i53 or i53-DM (DM). An optimal ssODN donor targeting GFP (GFP-1) was used in MCF10a cells whereas a suboptimal donor (GFP-2) was used in 293T cells. Donor and Cas9 RNP were then nucleofected and 96 h later, cells were analysed for GFP and BFP expression. Individual paired experiments are presented. c, Representative scatter plots for one of the biological replicates of the experiment shown in Fig. 4e and panel b (left graph). d, e, Representative agarose gels of the RFLP analysis for the experiments shown in Fig. 4f and Fig 4g. f, Quantitation of indels by TIDE analysis in the experiments shown in Fig 4f and 4g (N=4 for 293T cells, N=3 for K562 cells).
Supplementary Figure 8 CtIP promotes HDR with ssODN donors and i53 does not promote sister chromatid exchanges.
a, Representative scatter plots for one of the biological replicates of the experiment shown in Fig. 4h. b, Control immunoblot for the CtIP depletion in the experiment shown in Fig. 4h. c, U2OS EJ2-GFP cells were transfected with the vectors expressing Flag-tagged i53, its DM mutant or an empty vector control (EV) along with an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-transfection for each condition. Individual experiments are presented along with the mean +/− s.e.m., N=4. d, Control immunoblot for the experiment shown in (c). e, i53 does not impact SCE frequency. Representative micrographs DAPI-stained metaphases from HeLa cells transduced with AAVs encoding GFP, i53 or i53-DM and treated for 48h with BrdU and then either left untreated or treated with mitomycin C (MMC). f, Quantification of SCEs/chromosome; Each data point is one metaphase derived from 3 independent experiments, overlaid is a box-and-whisker plot (min-to-max values) with the bar at median and bounds at interquartile range. g, FLAG and tubulin immunoblots of whole cell extracts from HeLa cells transduced with the indicated viruses as controls for the experiment shown in panel f-g. h, Control experiment to assess inhibition of 53BP1 focus formation in cells transduced with the indicated AAV. Shown are the percentage of cell showing >5 53BP1 irradiation-induced DNA damage foci following 5 Gy irradiation. Individual data points are shown along with the means +/− s.d. (N=3). Please note that the blots shown are cropped and the uncropped versions can be found as Supplementary Information.
Supplementary Figure 9 Uncropped scans of gels.
Uncropped scans of gels using in this paper. Shown also are the molecular weight markers (all kDa). Dashed lines delineate two blots that were scanned at the same time or two sets of antibodies that were used simultaneously on the same blot.
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Canny, M., Moatti, N., Wan, L. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR–Cas9 genome-editing efficiency. Nat Biotechnol 36, 95–102 (2018). https://doi.org/10.1038/nbt.4021
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