A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells

Christopher A Vakulskas, Daniel P Dever, Garrett R Rettig, Rolf Turk, Ashley M Jacobi, Michael A Collingwood, Nicole M Bode, Matthew S McNeill, Shuqi Yan, Joab Camarena, Ciaran M Lee, So Hyun Park, Volker Wiebking, Rasmus O Bak, Natalia Gomez-Ospina, Mara Pavel-Dinu, Wenchao Sun, Gang Bao, Matthew H Porteus, Mark A Behlke

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Abstract

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.

OriginalsprogEngelsk
TidsskriftNature Medicine
Vol/bind24
Nummer8
Sider (fra-til)1216-1224
Antal sider9
ISSN1078-8956
DOI
StatusUdgivet - 2018

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