基因组编辑
干细胞
核糖核蛋白
造血
祖细胞
清脆的
生物
基因
Cas9
细胞生物学
遗传学
计算生物学
核糖核酸
作者
Christopher A. Vakulskas,Daniel P. Dever,Garrett R. Rettig,Rolf Turk,Ashley M. Jacobi,Michael A. Collingwood,Nicole M. Bode,Matthew 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
出处
期刊:Nature Medicine
[Springer Nature]
日期:2018-08-01
卷期号:24 (8): 1216-1224
被引量:596
标识
DOI:10.1038/s41591-018-0137-0
摘要
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. A bacterial screen yields a Cas9 variant that retains high on-target activity when delivered in the RNP format. As proof of principle, this Cas9 variant enables high-level correction of the sickle cell disease mutation in patient-derived HSPCs.
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