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

a, Gene targeting efficiency at the LMNA locus^{12, 19} 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 locus²⁰ 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 locus²² 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)²⁶ 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^#)²⁶ 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).