Translation of the CRISPR–Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when 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, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.
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We thank S. Mantri for collecting and purifying the HSPCs. We thank K. Lennox for critical review of the manuscript. The Band3 APC antibody was a kind gift from A. Narla and M. Narla (Stanford University). M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research and NIH grant support R01-AI097320 and R01-AI120766. G.B. acknowledges support from the Cancer Prevention and Research Institute of Texas (RR14008 and RP170721). We thank the Binns Program for Cord Blood Research at Stanford University for cord-blood-derived CD34+ HSPCs and also for SCD-HSPCs. Patients with SCD consented to the use of CD34+ HSPCs for research with the accompanying IRB approval.
M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input or opinions on the subject matter described in this manuscript. C.A.V., G.R.R., R.T., A.M.J., M.A.C., N.M.B., M.S.M., S.Y., and M.A.B. are employees of Integrated DNA Technologies (IDT), which sells reagents similar to some described in the manuscript.
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Vakulskas, C.A., Dever, D.P., Rettig, G.R. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med 24, 1216–1224 (2018). https://doi.org/10.1038/s41591-018-0137-0
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