Figure 2.

Excess sgRNA in RNP Formation Leads to Efficient Editing in CD34⁺ HSPCs, Compensating for the Lack of an Alt-R EE

(A and B) RAG2- (A) and RAG1 (B)-targeting gRNAs were used as Alt-R 2-part XT gRNA or Alt-R sgRNA in CD34⁺ HSPCs. RNPs precomplexed at a 1:1.2 Cas9:gRNA molar ratio were electroporated into the cells with an Alt-R EE (left). Alternatively, RNPs were precomplexed at an elevated 1:2.5 Cas9:gRNA molar ratio and delivered to the cells without an Alt-R EE (right). Either 1 μM or 4 μM RNP concentrations (light and dark bars, respectively) were examined, and indel-editing frequencies at the on-target sites were analyzed by NGS. Bars represent mean indel-editing percentages ± SEM (from three independent human donors), and editing percentages are designated above the bars. Mock-electroporated negative controls were used to subtract background indels when performing the analyses.