A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells
By Christopher A. Vakulskas, D. Dever, G. Rettig, R. Turk, Ashley M. Jacobi, Michael A. Collingwood, Nicole M. Bode, Matthew S. McNeill, Shuqi Yan, J. Camarena and 10 more. Nature Medicine, 2018
671 citations recorded by the source corpus.
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.
Subfields: Genetics, Molecular Biology, Public Health, Environmental and Occupational Health
View Paper: https://www.semanticscholar.org/paper/e5e18da9b2f4540ec20006350ccf770ad7fe42bb
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Citations in the corpus
- CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “for the DNA:RNA heteroduplex to induce DSBs, thereby reducing OTEs while maintaining editing efficacy (38, 39).”
- CRISPR technology: A decade of genome editing is only the beginning (2023, methodology): “To reduce the offtarget effects of CRISPR-Cas nucleases resulting from unintended binding and cleavage, researchers have utilized a combination of rational design and selection to develop highfidelity Cas variants [such as SpCas9-HF1 (101), evoCas9 (102), HiFiCas9 (103)] and the Cas9_ R63A/Q768A var”