Figure 5.

Experimental outline of Cas9 variant gene editing in HSPCs. (A) Experimental overview of electroporation of Cas9 variants delivered as IVT mRNA to CD34⁺ HSPCs. Cas9 variants were electroporated at equimolar amounts (3 pmol) with 120 pmol of IVT sgRNA targeting the SCD mutation at the HBB locus, and either a ssODN (3 μM) or AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. (B) Viability of CD34⁺ HSPCs edited with Cas9 variants, and an ssODN or AAV6 donor 24 h post-electroporation. n = 2–6 biological replicates. Center line represents mean. Differences are not significant if not specified, based on Wilcoxon rank sum test.