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

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

来源：Nature Medicine, 2018, 24(8): 1216-+.

DOI：10.1038/s41591-018-0137-0

• OFF-TARGET
• MARROW TRANSPLANTATION
• CRISPR-CAS9 NUCLEASES
• MESSENGER-RNA
• BONE-MARROW
• T-CELLS
• GENOME
• CRISPR/CAS9
• INTEGRATION
• MUTATION

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.

• 出版日期2018-8