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

Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, Bode NM, McNeill MS, Yan S, Camarena J, Lee CM, Park SH, Wiebking V, Bak RO, Gomez-Ospina N, Pavel-Dinu M, Sun W6, Bao G, Porteus MH, Behlke MA
Source: Nat Med
Publication Date: (2018)
Issue: 24(8): 1216-1224
Research Area:
Immunotherapy / Hematology
Stem Cells
Gene Expression
Regenerative medicine
Cells used in publication:
Species: human
Tissue Origin: kidney
T cell, human stim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® 96-well Systems
4D-Nucleofector® X-Unit

Cell Culture stimulated T-cells: Cells were cultured at 37 °C, 5% CO2 in X-Vivo 15 (Lonza) supplemented with 5% human serum (Sigma-Aldrich, St. Louis, MO, USA), and 100 IU/mL human recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA) and 10ngmL-1 human recombinant IL-7 (BD Biosciences, San Jose, CA, USA). T cells were activated using immobilized anti-CD3 (clone OKT3, Tonbo Biosciences) and soluble anti-CD28 (clone CD28.2, Tonbo Biosciences) for 3 d before electroporation. 

Nucleofection: stimulated T cells: Cells were resuspended in electroporation solution, mixed with the precomplexed RNP and electroporated using a 4D-Nucleofector (Lonza) using program EO-115. To assess homologous recombination, cells were transduced with rAAV after electroporation. CD34+: All Cas9 proteins (WT and mutants) were obtained from Integrated DNA Technologies. RNP complexes were made by mixing Cas9 with sgRNA at a molar ratio of 1:2.5 (Cas9:sgRNA) at 25 °C for 10 min before electroporation. CD34+ cells were electroporated with RNP at a concentration of 300 µ g mL-1 (unless stated otherwise). CD34+ cells were
resuspended in either P3 buffer (Lonza) or buffer 1M67 and electroporated using the Lonza 4D Nucleofector (with program DZ100). Cells were plated at 250,000– 500,000 cells/mL in cytokine-supplemented medium, and then rAAV6 was delivered onto cells immediately (within 15 min) upon plating after electroporation
ranging vector genomes per cell from 50,000–100,000 as titered by qPCR or 5,000–10,000 as titered by droplet digital PCR (ddPCR). HEK 293 screening has been performed in the 96Well Shuttle system.


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.