Fig. 5: Generation of heterozygous mutations requires protection of one allele.

a Target sequence in the GFP reporter, and ssODN repair templates to create a missense Y66H mutation (ssODN M), a silent T65T blocking mutation (ssODN B) or no mutation (ssODN W). HDR repair of ssODN B T65T results in a protected GFP (pGFP) allele. Cells bearing unmodified or pGFP alleles are indistinguishable by FACS and are scored cumulatively as GFP. b GFP editing with ssODN M, B or W individually, or in M + B and M + W combination, and effect on DNA repair outcome frequency under cold shock condition. HDR* indicates the frequency of heterozygous double-positive BFP/GFP or compound heterozygous double-positive BFP/pGFP repair outcomes. GFP-positive cells include unmodified cells and HDR-mediated monoallelic pGFP/GFP, pGFP/indel (Δ), or biallelic pGFP/pGFP repair outcomes. c Distribution of biallelic repair outcomes shown in b. d Biallelic BFP/GFP repair outcome frequency measured in c. e Representative FACS plots of BFP conversion only (ssODN M) or heterozygous compound BFP/pGFP mutations (ssODN M + B). All data are presented as the mean ± S.D. of three technical replicates of independent electroporations for each respective condition. Source data are provided as a Source Data file.