Fig. 2

Nuclease-mediated HDR is associated with predominant indels leading to impaired UROS functionality. a (Left) Scheme of SacI-digested PCR products obtained for alleles with or without HDR. (Center) Illustrative RFLP analysis of non-transfected HEK293T cells (NT) or transfected with nuclease only or co-delivered with the 181nt-ssODN template. (Right) HDR frequency induced by nuclease and a 181nt-ssODN (n = 5). b (Left) NGS analysis of allelic outcomes and associated HDR/indel ratio following transfection of HEK293T cells with nuclease and 181nt-ssODN. (Center) Frequencies of reads carrying either insertion or deletion. Region spanning sgRNA sequence is highlighted in grey. (Right) Most common observed alleles (with frequencies ≥ 1%) aligned on the sgRNA sequence. HDR modification in blue and indels in red. c (Left) Quantification of UROS-specific activity from HEK293T cells NT or transfected with nuclease and 181nt-ssODN (n = 3). Values are normalized with NT cells. (Right) Fluorocyte frequencies and illustrative flow cytometry results from NT or HEK293T cells transfected with nuclease and a 181nt-ssODN (n = 5). Blue and red dots (and associated percentages) depict non-fluorescent cells and fluorocytes, respectively, with type-I porphyrin accumulation. Results are presented as mean ± SEM. The data are from independent experiments. Statistical significance is inferred on raw data using two-tailed unpaired t test for UROS-specific activity. ***p < 0.001. For a, c, source data are provided as a Source data file