Journal Article

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

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

2018

Overview

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.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/61/201/2110 / 692/308/2171 / 692/699/1541/13 / Anemia, Sickle Cell - genetics / Anemia, Sickle Cell - therapy / Antigens, CD34 - metabolism / Bacterial genetics / Base Sequence / Biomedical and Life Sciences / Biomedicine / Cancer Research / Care and treatment / CCR5 protein / CD34 antigen / Cells (biology) / CRISPR / CRISPR-Associated Protein 9 - genetics / Efficiency / Escherichia coli / Fidelity / Gene Editing / Gene mutation / Gene targeting / Genes / Genetic aspects / Genetic modification / Genome editing / Genomes / Genomics / Health aspects / HEK293 Cells / Hematopoietic stem cells / Hematopoietic Stem Cells - metabolism / Humans / Infectious Diseases / Lymphocytes / Lymphocytes T / Medical schools / Metabolic Diseases / Molecular Medicine / Mutation / Mutation - genetics / Neurosciences / Plasmids / Point mutation / Progenitor cells / Ribonucleoproteins - metabolism / Sickle cell anemia / Sickle cell disease / Stem cells / T cells / Therapeutics