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Nat Med. 2018 Aug;24(8):1216-1224. doi: 10.1038/s41591-018-0137-0. Epub 2018 Aug 6.

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

Author information

1
Integrated DNA Technologies, Inc., Coralville, IA, USA.
2
Department of Pediatrics, Stanford University, Stanford, CA, USA.
3
Department of Bioengineering, Rice University, Houston, TX, USA.
4
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
5
Aarhus Institute of Advanced Studies (AIAS), Aarhus University, Aarhus, Denmark.
6
Biomaterials and Advanced Drug Delivery Laboratory, Stanford University School of Medicine, Palo Alto, CA, USA.
7
Department of Pediatrics, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.
8
Integrated DNA Technologies, Inc., Coralville, IA, USA. mbehlke@idtdna.com.

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.

PMID:
30082871
PMCID:
PMC6107069
DOI:
10.1038/s41591-018-0137-0
[Indexed for MEDLINE]
Free PMC Article

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