Extended Data Fig. 10: Assessment of off-target editing in vitro and in vivo.
a. Thirty-eight potential genomic sgRNA-25 off-target sites in total were evaluated by amplicon deep sequencing and quantified with CRISPResso2. To assess potential off-target effects, we examined the editing positions targeting A5 (OT1–7, OT9–14, OT17–18, OT21, OT25, OT29–38), A6 (OT8, OT15, OT19–20, OT22, OT23–24, OT26–28), and A15 (OT16), which were identified as mutation hotspots. For all sites, the off-target sites with difference in edit frequency between mock and edited samples of less 0.15%. b. Ten potential genomic sgRNA-25 off-target sites were evaluated by amplicon deep sequencing in BM cells from engrafted mice after LNP-028-ABE8e-HBG injection. For all sites, the off-target sites with difference in edit frequency between mock and edited samples of less 0.12%. c. HBG ABE8e cleavage sites are indicated by inverted triangles. Quantitative PCR (qPCR) primers targeting the intergenic sequence between the cleavage sites are indicated (red arrows). The larger deletion presumed to arise from simultaneous ssDNA breaks in HBG2 and HBG1, with the loss of the intervening 4.9-kb, is shown (dotted line). Primers for Δ 366-bp denote a quantitative qPCR assay that detects deletions ≥366-nt upstream of the cleavage site in HBG1. d. The analysis of large fragment deletions was conducted following the electroporation of ABE8e RNP and Cas9 RNP in human CD34+ cells. e. Five days post ABE8e RNP editing, base editing efficiency in CD34+ cells were examined using deep sequence analysis. f. Five days post Cas9 RNP editing, indel frequency in CD34+ cells were analyzed by synthego. g. β-like globin gene expression was analyzed by RT-qPCR in erythroid cells differentiated in vitro from ABE8e and Cas9 RNP-edited CD34+ HSPCs. All data are representative of three biologically independent replicates. All statistical significances in the figures were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test, and data represent mean ± SD. Statistical analysis showed p < 0.0001 for sgRNA-25, sgRNA-35, and sgRNA-41 in ABE8e RNP groups compared with Cas9 RNP controls, as shown in panels d and g.
