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

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.

Clinical applications of CRISPR‐based genome editing and diagnostics

“…This risk is mitigated in the CRISPR clinical trials that are under way, as a subset of the cells edited ex vivo will be analyzed for genome‐wide OTEs before autologous transplantation of the edited cells into the patient. Furthermore, variants of Cas enzymes are being engineered with higher fidelity, which could limit OTEs to levels that are not of clinical concern . The recent observation that Cas9 is capable of infrequently inducing larger genomic rearrangements stimulated significant apprehension .…”

“…Furthermore, variants of Cas enzymes are being engineered with higher fidelity, which could limit OTEs to levels that are not of clinical concern. 106,107 The recent observation that Cas9 is capable of infrequently inducing larger genomic rearrangements stimulated significant apprehension. 108 Such events may be cell type-specific and could be preventable by using genomeediting approaches that do not induce double-strand breaks, such as nickases 109,110 or base editors.…”

Advances in the gene therapy of monogenic blood cell diseases

“…These RNPs have been implemented with the design of gRNAs aiming at increasing their stability, thus achieving high HDR efficiencies in HSPCs . These new RNPs together with the use of AAV6 for the delivery of donor templates are thus approaching the field of gene editing to the treatment of patients with hematological disorders . To reduce the complexity of gene editing—for example, by avoiding handling of viral vectors—the co‐delivery of nucleases together with ssODN donors has been used to correct specific mutations, including the SCD mutation in HSCs …”

“…Different approaches, such as GUIDE‐seq or CIRCLE‐seq have been developed to facilitate the identification of off‐targets in the human genome. Furthermore, different refinements in gene‐editing strategies have been established to reduce the generation of off‐targets, including paired Cas9 nickases, or high‐fidelity Cas9 nucleases …”

“…155,171 These new RNPs together with the use of AAV6 for the delivery of donor templates are thus approaching the field of gene editing to the treatment of patients with hematological disorders. 156,172,173 To reduce the complexity of gene editing-for example, by avoiding handling of viral vectors-the co-delivery of nucleases together with ssODN donors has been used to correct specific mutations, including the SCD mutation in HSCs. 155,165 Since non-homologous end joining (NHEJ) is the most efficient mechanism for the repair of DSBs, particularly in non-dividing cells, 174,175 this strategy has been used in the first FDA approved gene editing trials with autologous HSCs.…”