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

Abstract

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). 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 cells 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 variants 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.

Figure 1. Identification of 53BP1-binding ubiquitin variants

a, Schematic representation of 53BP1, highlighting the focus-forming region (FFR), which is necessary and sufficient for the recruitment of 53BP1 to DSB sites. b, Phage enzyme-linked immunosorbent assays (ELISAs) for binding to the following immobilized proteins (color coded as indicated in the panel): USP5, USP7, SMURF1, HACE, HOIP, HOIL, 53BP1 (Tudor-UDR region), NBD, SMURF2, CDC4, OTUB1, FBW7, USP8, ITCH, USP21, USP14 and BSA. Bound phages were detected spectrophotometrically (optical density at 450 nm), and background binding to neutravidin was subtracted from the signal. c, Sequence alignments of the 53BP1-binding Ubvs. d, Pulldown assays of the indicated GST-Ubv fusion with either MBP alone (−) or MBP fused to the Tudor or Tudor-UDR fragments of 53BP1. The asterisk (*) labels bands that we attribute as possible protein degradation products. e, the various MBP proteins used in the pulldown assays were separated by SDS-PAGE and stained with Coomassie brilliant blue. f, Competition assay in which the GST-UbvG08 was prebound to the MBP-Tudor fusion of 53BP1. Increasing amounts of a synthetic peptide derived from the region of H4K20me2 were added. After extensive washing, bound proteins were analyzed by immunoblotting against GST and MBP. g, Isothermal titration calorimetry profiles obtained by titration of UbvG08 (squares) or UbvG08-DM (circles) titrated into a solution of the 53BP1 Tudor protein. Curves were fitted with a one-set-of-sites model. The dissociation constant (K_d) for the UbvG08-53BP1 interaction is indicated (N=3). Please note that the gels and blots shown in panels d,e,f are cropped and uncropped version can be found as Supplementary Information.

Figure 2. Structure of the UbvG08 bound to the 53BP1 Tudor domain

a, Ribbons representation of the UbvG08 (shown in green) – 53BP1 Tudor domain (shown in gold) complex. The hydrophobic patch centered on I44 of the UbvG08 structure is highlighted in red. b, Reciprocal interaction surfaces on UbvG08 (top) and 53BP1 Tudor domain (bottom). Contact residues are highlighted on their respective surfaces. c, Zoom-in of the UbvG08-53BP1 Tudor domain contact region. Hydrogen and salt interactions are denoted by black dotted lines. d, MBP pulldown assay of GST fused to ubiquitin (Ub) or to the indicated UbvG08 proteins, with the MBP-53BP1-Tudor protein. e, MBP-pulldown assay of GST fused to UbvG08, its L70V mutant or the indicated Ub proteins, with the MBP-53BP1-Tudor protein. f, MBP-pulldown assay of GST fused to ubiquitin (Ub) or to the indicated UbvG08 proteins, with the MBP-53BP1-Tudor protein. PD, pulldown. IB, immunoblot. Please note that the blots shown in panels d,e,f are cropped and uncropped version can be found as Supplementary Information.

Figure 3. The i53 protein inhibits 53BP1 and activates gene conversion

a–b, U2OS cells were transfected with vectors expressing i53, its 53BP1-binding deficient mutant (DM) or an empty vector (EV) control. Cells were then X-irradiated with a 10 Gy dose and processed for immunofluorescence with the indicated antibodies 1 h post-irradiation (IR). DAPI staining (not shown) was used to delineate the outline (dashed lines) of the cell nuclei. The region in the magnified inset is indicated with a square. Quantitation of the experiment is shown in panel (a) where each circle is a biological replicate and the bar is at the mean (N=3 for EV, N=4 for i53 and i53-DM), whereas in (b) representative micrographs are shown. Arrowheads indicate Flag-positive cells. Additional micrographs are shown in Supplementary Fig. 3a. c, Parental or 53BP1ΔU2OS cells transfected with vectors expressing i53, the DM mutant or an empty vector (EV) control were irradiated (2 Gy) 1 h before being processed for immunofluorescence. Cell cycle stage was assessed by Cyclin A staining. Each circle represents a biological replicate and the bar is at the mean; N=4 (U2OS) and N=3 (53BP1ΔU2OS). Micrographs are shown in Supplementary Fig. 3b. d, Immunoprecipitation (IP) of Flag-tagged proteins from extracts prepared from 293T cells transfected with vectors expressing Flag-i53 or the i53-DM mutant. Proteins were separated by SDS-PAGE and immunoblotted (IB) for Flag and 53BP1. e, Schematic of the DR-GFP assay. f. U2OS DR-GFP were first transfected with siRNAs targeting the 53BP1 or BRCA1 mRNAs along with a non-targeting siRNA (CTRL). 24 h post-transfection, cells were transfected with the I-SceI expression vector and the percentage of GFP-positive cells was determined 48 h post-I-SceI transfection for each condition. The values were normalized to the CTRL siRNA condition. Each point is a biological replicate and the bar is at the mean ± s.e.m; N=4. g, U2OS DR-GFP cells were transfected with the vectors expressing i53, the 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 and was normalized to the empty vector condition. Each point is a biological replicate and the bar is at the mean ± s.e.m; N=4. h, U2OS DR-GFP cells were transfected with either an empty vector (EV) or vectors expressing Flag-tagged i53 or the DM mutant along with an I-SceI expression vector. Cells were treated either with DMSO (−) 1 µM SCR7 or 1 µM of the SCR7 pyrazine analog (pyrSCR7). The percentage of GFP-positive cells was determined 48 h post-transfection for each condition and was normalized either to the EV (left) or DM (right) conditions. Each point is a biological replicate and the bar is at the mean ± s.e.m (N=3 for all experiments on the left graph, except for SCR7+DM and SCR7+i53 where N=2; N=4 for all experiments on the right graph). Please note that the blots shown in panel d are cropped and uncropped version can be found as Supplementary Information.

Figure 4. Stimulation of HDR by i53 with dsDNA and ssODN donors

a, Gene targeting efficiency at the LMNA locus^, in parental or 53BP1Δ U2OS cells following transfection with vectors expressing Flag-tagged i53 or its DM mutant or an empty vector control (EV). The DNA-PK inhibitor NU7441 was also added where indicated. 24 h post-transfection, cells were analysed for mClover fluorescence. Individual experiments are presented along with the mean +/− s.d., (N=3). b, Mono- and bi-allelic gene targeting at the HIST1HB2K locus in K562 cells previously transduced with AAV coding for i53 or i53-DM (DM). The HIST1HB2K-mAG and HIST1HB2K-mCherry donors were introduced by nucleofection at the same time as a Cas9 RNP targeting HIST1HB2K. Control reactions where 72 h post-transfection, cells were analysed for mAG and mCherry fluorescence. Individual experiments are presented along with the mean +/− s.d., (N=3). c, Gene targeting efficiency at the Hsp90a1 locus in mouse embryo fibroblasts previously transduced with AAV coding for i53 or i53-DM (DM). The Hsp90a1-t2A-ZsGreen template vector was introduced by transfection at the same time as the vector coding for the sgRNA and Cas9. 8 days post-transfection, cells were analysed for ZsGreen fluorescence. Individual experiments are presented along with the mean +/− s.d., (N=3). The no sgRNA controls were only done once. d, HDR at the CCR5 locus in K562 cells nucleofected with ZFN mRNA and an XhoI inserting CCR5 plasmid donor (CCR5*), followed by nucleofection 24 h later with increasing concentrations of i53 mRNA or i53-DM (DM) mRNA, or left untreated (control). 24 h later, cells were collected and gene targeting was determined by RFLP analysis. Individual experiments are presented along with the mean +/− s.d., (N=3). e, BFP-to-GFP conversion by ssODN-mediated HDR in 293T cells previously transduced with AAV coding for i53 or i53-DM (DM). An optimal ssODN donor targeting GFP (GFP-1) and Cas9 RNP were then nucleofected and 96 h post-transfection, cells were analysed for GFP and BFP expression. Individual paired experiments are presented (N=4). f, g, HDR at the CCR5 and CXCR4 loci in 293T (f) and K562 (g) cells previously transduced with AAV coding for i53 or i53-DM (DM). ssODN donor stargeting CCR5 (CCR5^#) or CXCR4 (CXCR4^#) and Cas9 RNP were then nucleofected and 72 h post-transfection, editing was determined by RFLP analysis. Individual paired experiments are presented on the left graphs whereas the relative editing efficiency is presented in the right graphs where data is presented as the mean +/− s.d., (N=4 for f and N=3 for g). h, BFP-to-GFP conversion by ssODN-mediated HDR in 293T cells was carried out as panel e with the exception that cells were either transfected with a non-targeting siRNA (siCTRL), a pool of siRNAs targeting CtIP (siCtIP (pool)) or a different, custom-designed siRNA targeting CtIP (siCtIP-5). Individual experiments are presented along with the mean +/− s.d., (N=4 for siCTRL, N=3 for siCTIP conditions).