Figure 4.

Precise gene correction by HDR using high-fidelity RNPs

(A) U2OS cells were electroporated with WT SpCas9, HiFi Cas9, or rCas9HF RNPs targeting the EMX1, BCR, and HBB loci together with specific ssODN templates (Table S1). Indel formation (NHEJ) and HDR events were quantified at 3 days post-transfection. (B) HDR/indel ratios calculated for the EMX1, BCR, and HBB loci are from (A). (C) Quantification of HDR-mediated correction in the CFTR locus targeting two clinically relevant mutations causing cystic fibrosis. HEK293 cells stably expressing mutated CFTR minigenes were electroporated with WT SpCas9, HiFi Cas9, or rCas9HF RNP complexes and an ssODN donor. Indel frequencies and HDR levels were analyzed 3 days post-electroporation. (D) HDR/indel ratios calculated for the two CFTR loci are from (C). Data reported as mean ± SEM for n ≥ 2 biologically independent replicates; statistical significance was assessed using one-way ANOVA. Bars in (A) and (C) are superimposed.