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Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR–Cas9 genome-editing efficiency

Nature Biotechnology volume 36, pages 95–102 (2018)Cite this article

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Abstract

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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Figure 1: Identification of 53BP1-binding ubiquitin variants.
Figure 2: Structure of the UbvG08 bound to the 53BP1 Tudor domain.
Figure 3: The i53 protein inhibits 53BP1 and activates gene conversion.
Figure 4: Stimulation of HDR by i53 with dsDNA and ssODN donors.

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  • 14 December 2017

    In the version of this article initially published, the affiliation footnotes of Andrew Vorobyov and Sachdev S Sidhu were erroneously given as number 4, the Skirball Institute of Biomolecular Medicine, instead of number 5, The Donnelly Centre for Cellular and Biomolecular Research. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

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.).

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Author notes

  1. Amélie Fradet-Turcotte, Alexandre Orthwein & Andreas Ernst

    Present address: Present addresses: St-Patrick Research Group in Basic Oncology, Université Laval Cancer Research Center, and Oncology Axis-CHU de Québec Research Center – Université Laval, Quebec City, Quebec, Canada (A.F.-T.); Department of Oncology, McGill University, Montreal, Quebec, Canada and Segal Cancer Center, Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada (A.O.); and Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany (A.E.).,

  2. Marella D Canny and Nathalie Moatti: These authors contributed equally to this work.

Authors and Affiliations

  1. The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Marella D Canny, Nathalie Moatti, Leo C K Wan, Amélie Fradet-Turcotte, Michal Zimmermann, Alexandre Orthwein, Yu-Chi Juang, Sylvie M Noordermeer, Marcus D Wilson, Meagan Munro, Timothy F Ng, Tiffany Cho, Frank Sicheri & Daniel Durocher

  2. Department of Molecular Genetics, University of Toronto, Ontario, Canada

    Leo C K Wan, Timothy F Ng, Tiffany Cho, Sachdev S Sidhu, Frank Sicheri & Daniel Durocher

  3. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Danielle Krasner, Eduardo Seclen & Paula M Cannon

  4. Department of Cell Biology, Skirball Institute of Biomolecular Medicine, NYU Langone Medical Center, New York, New York, USA

    Pedro A Mateos-Gomez

  5. Banting and Best Department of Medical Research, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Ontario, Canada

    Wei Zhang, Andrew Vorobyov, Andreas Ernst & Sachdev S Sidhu

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Contributions

M.D.C. generated the mutations for the analysis by pull-down and carried out most of the pull-down experiments, generated the protein crystals, conducted the ITC experiments and carried out some of the initial assays demonstrating efficacy of i53. N.M. carried out the DR- and EJ2-GFP assays, all assays with AAV-i53 in human cells along with all ssODN assays and the determination of 53BP1 foci in G1 cells. L.C.K.W. refined and analyzed the crystal structure and prepared the figures describing the structure. A.F.-T. produced the GST-53BP1 protein for Ubv selection and the GST-fusion Ubv proteins for the initial pull-down assays she carried out. Y.-C.J. supervised the crystallization and collected the diffraction data. A.O. carried out the LMNA gene targeting assays with the help of T.F.N. W.Z. carried out the Ubv selections with A.V. and A.E. D.K. and E.S. conducted HDR analyses in K562 cells. T.C. assessed the impact of i53 on p53 to answer a reviewer comment whereas M.Z. tested the impact of i53 of SCE rates. P.M.C. supervised D.K. and E.S. P.A.M.-G. performed mouse gene targeting assay. S.M.N. carried out the PARP inhibitor assays using cells generated by M.Z. M.D.W. helped with biochemical experiments. M.M. did the immunoprecipitation mass spectrometry and helped M.D.C., A.F.-T. and A.O. S.S.S. supervised W.Z., A.V. and A.E. F.S. supervised L.C.K.W. and Y.-C.J. D.D. conceived and supervised the project, obtained funding and wrote the manuscript with L.C.K.W., M.D.C. and F.S., with input from the other authors.

Corresponding author

Correspondence to Daniel Durocher.

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Competing interests

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)

a, Representative micrographs of the experiment shown in Fig. 3a-b. b, Representative micrographs of the experiment shown in Fig. 3c.

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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