Figure 5.

Molecular analysis of treated X-CGD HSPCs at on-target (ON) and OT sites confirm the high specificity of the SpCas9/sg/ODN genome-editing system maintained in the presence of i53. (A) Long-range PCR at CYBB E7 target site in HSPCs (naive, Cas9/sg alone, or with i53, ODN donor, or both). Genomic deletions shown by loss of coverage of sequencing reads. (B) Graph summarizing frequency of large deletions in samples treated as indicated. (C) ON and OT sites identified by circularization for high-throughput analysis of nuclease genome-wide effects by sequencing (CHANGE-seq) with mismatches as shown, with the number of reads for respective sites as indicated on the right. (D) Manhattan plot of the ON and OT sites with the sg7 sgRNA on CYBB c.676 C>T mutation X-CGD patient genomic DNA. The arrow indicates CYBB on chromosome X. (E) Indel frequency at ON and OT sites before (input) and after transplant (26 weeks) in NSG (output) of HSPCs (naive [gray]), GE with (blue) or without (red) i53 (mean ± standard deviation, ordinary one-way analysis of variance, not significant). (F) Percentages of reads with size discrepancies indicating indels for naive (N), or treated with (+) or without (-) i53 at ON and at each OT site. (G) Frequency of DSB repair events in vitro (IV) and posttransplant (fresh BM) with/without i53.