Journal
NATURE MEDICINE
Volume 24, Issue 8, Pages 1216-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41591-018-0137-0
Keywords
-
Funding
- Amon Carter Foundation
- Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research
- NIH [R01-AI097320, R01-AI120766]
- Cancer Prevention and Research Institute of Texas [RR14008, RP170721]
Ask authors/readers for more resources
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, CCRS, 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available