Engineered novel high-fidelity Cas9 delivered as a ribonucleoprotein complex enables high frequency gene editing in human haematopoietic stem and progenitor cells

Translation of the CRISPR/Cas9 system to human therapeutics holds high promise.Specificity remains a concern, however, especially when modifying stem cellpopulations. We show that existing rationally-engineered Cas9 high fidelity variantshave reduced on-target activity using the therapeutically relevant ribonucleoprotein(RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolatevariants that retain activity in the RNP format. Introduction of a single point mutation,R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFiCas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci(HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem andprogenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Valmutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wideutility for both basic science and therapeutic genome editing applications.