BioProject

Specificity remains a concern, however, especially when modifying stem cell

populations. We show that existing rationally-engineered Cas9 high fidelity variants

have reduced on-target activity using the therapeutically relevant ribonucleoprotein

(RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate

variants 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. HiFi

Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci

(HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and

progenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9

mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Val

mutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wide

utility for both basic science and therapeutic genome editing applications. Less...