1. Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins.
- Author
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Hiatt J, Cavero DA, McGregor MJ, Zheng W, Budzik JM, Roth TL, Haas KM, Wu D, Rathore U, Meyer-Franke A, Bouzidi MS, Shifrut E, Lee Y, Kumar VE, Dang EV, Gordon DE, Wojcechowskyj JA, Hultquist JF, Fontaine KA, Pillai SK, Cox JS, Ernst JD, Krogan NJ, and Marson A
- Subjects
- Animals, Humans, Mice, CRISPR-Cas Systems genetics, Genome genetics, Myeloid Cells metabolism, Ribonucleoproteins metabolism
- Abstract
Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14
+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies., Competing Interests: Declaration of interests The authors declare competing interests: T.L.R. is a co-founder of Arsenal Biosciences. A.M. is a compensated co-founder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. was a compensated member of the scientific advisory board at PACT Pharma and was a compensated advisor to Juno Therapeutics and Trizell. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, and PACT Pharma. A.M. has received honoraria from Merck and Vertex, a consulting fee from AlphaSights, and is an investor in and informal advisor to Offline Ventures. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. A.M., T.L.R., and E.S. are holders of patents pertaining to, but not resulting from, this work. The Krogan laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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