A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.
Nat Med
; 24(8): 1216-1224, 2018 08.
Article
em En
| MEDLINE
| ID: mdl-30082871
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.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Ribonucleoproteínas
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Células-Tronco Hematopoéticas
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Edição de Genes
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Proteína 9 Associada à CRISPR
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Mutação
Limite:
Humans
Idioma:
En
Revista:
Nat Med
Assunto da revista:
BIOLOGIA MOLECULAR
/
MEDICINA
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos