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


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2018-08

AUTHORS

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

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. More... »

PAGES

1216-1224

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/s41591-018-0137-0

    DOI

    http://dx.doi.org/10.1038/s41591-018-0137-0

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1105928907

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/30082871


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