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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.

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Fig. 1: On-target activity of previously described high-fidelity Cas9 mutants delivered as RNPs in human cells.
Fig. 2: Unbiased bacterial selection for cas9 mutants that reduce off-target editing and maintain on-target potency.
Fig. 3: The HiFi Cas9-R691A mutant maintains on-target editing activity when delivered as a RNP.
Fig. 4: HiFi Cas9 globally reduces off-target activity with both stable Cas9 expression and RNP delivery.
Fig. 5: RNP HiFi Cas9 facilitates on-target editing potency near the WT level, with large OTE reductions in primary CD34+ HSPCs.
Fig. 6: HiFi Cas9 delivered as a RNP mediates robust p.E6V HBB gene correction in CD34+ SCD-HSPCs while significantly reducing off-target activity.

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Acknowledgements

We thank S. Mantri for collecting and purifying the HSPCs. We thank K. Lennox for critical review of the manuscript. The Band3 APC antibody was a kind gift from A. Narla and M. Narla (Stanford University). M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research and NIH grant support R01-AI097320 and R01-AI120766. G.B. acknowledges support from the Cancer Prevention and Research Institute of Texas (RR14008 and RP170721). We thank the Binns Program for Cord Blood Research at Stanford University for cord-blood-derived CD34+ HSPCs and also for SCD-HSPCs. Patients with SCD consented to the use of CD34+ HSPCs for research with the accompanying IRB approval.

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C.A.V. performed and designed the bacterial screen and subsequent identification of the HiFi mutants. C.A.V., M.A.C., and N.M.B. cloned and purified all proteins examined in this study. C.A.V., G.R.R., R.T., A.M.J., and M.S.M. performed NGS on-target and off-target editing experiments with RNP in human cells. C.A.V., M.A.C., and S.Y. created and characterized the HEK293 Cas9 stable cell lines. D.P.D., J.C., R.O.B., V.W., M.P.-D., and N.G.-O. carried out experiments related to HSPC and T cell gene editing. G.B., C.M.L., and S.H.P. carried out the NGS analysis of HSPC HBB editing events. W.S. carried out the HPLC analysis of hemoglobin tetramers. M.A.B. and M.H.P. directed the research and participated in the design and interpretation of the experiments and the writing of the manuscript. C.A.V., D.P.D., M.H.P., and M.A.B. wrote the manuscript with assistance from all authors.

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Correspondence to Matthew H. Porteus or Mark A. Behlke.

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M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input or opinions on the subject matter described in this manuscript. C.A.V., G.R.R., R.T., A.M.J., M.A.C., N.M.B., M.S.M., S.Y., and M.A.B. are employees of Integrated DNA Technologies (IDT), which sells reagents similar to some described in the manuscript.

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Vakulskas, C.A., Dever, D.P., Rettig, G.R. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med 24, 1216–1224 (2018). https://doi.org/10.1038/s41591-018-0137-0

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