Fig. 4: CDC7 inhibition increases HR and SSTR in primary human cells.

a XL413 increases HR at endogenous loci in T cells. Primary CD3⁺ T cells from two healthy blood donors were nucleofected with RNPs targeting three different genomic loci (RAB11A, TUBA1B, and CLTA) with a linear dsDonor template to knock-in a fluorescent reporter protein fused to the N-terminal end of the target protein. Nucleofected cells were treated with the indicated doses of XL413 for 24 h, then the percentage of reporter-positive T cells was quantified by flow cytometry 4 days after nucleofection. Gating strategy is depicted in (Supplementary Fig. 4a). b XL413 increases SSTR at endogenous loci. Primary CD3⁺ T cells were nucleofected with RNPs targeting the indicated loci with ssDonors to insert point mutations, treated with the indicated concentration of XL413 for 24 h. After 4 days, gDNA was extracted and SSTR and NHEJ frequencies determined by amplicon sequencing. c XL413 increases the frequency of SNP conversion. RNPs targeting the CD4 locus and an ssDonor encoding a naturally occurring SNP (Supplementary Fig. 4b–d) were introduced into T cells from three donors, and editing outcomes quantified by flow cytometry after 4 days. Statistical significances were calculated by ordinary one-way ANOVA and Dunnet’s multiple comparison test (adjusted p-values are reported as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). d XL413 increases the frequency of SNP conversion in the HBB gene in primary human CD34⁺ HSPCs. RNPs targeting the HBB locus and an ssDonor encoding the E6V mutation²⁵ were nucleofected into HSPCs in the presence or absence of 30 μM XL413, gDNA was extracted after 4 days, and mutation frequencies determined by amplicon sequencing. Statistical significances were calculated by unpaired two-tailed t-test using the Holm-Sidak method (p-values are reported as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). e XL413 increases the frequency of SSTR in primary human CD34⁺ HSPCs. Cells were nucleofected with RNPs targeting TOMM20 with a ssDonor that contains three synonymous SNPs in the presence or absence of 30 μM XL413 for 24 h, gDNA was extracted after 4 days, and SSTR and NHEJ frequencies were determined by amplicon sequencing. Values are shown as mean ± SD of the indicated number of samples. Source data are available in the Source Data file.