Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins
Devin A. Cavero,
Michael J. McGregor,
David E Gordon,
Jonathan M Budzik,
Kelsey M. Haas,
Mohamed S. Bouzidi,
Judd F Hultquist,
Jason A. Wojcechowskyj,
Krystal A Fontaine,
Satish K Pillai,
Jeffery S. Cox,
Joel D. Ernst,
Nevan J. Krogan,
Posted 15 Mar 2020
bioRxiv DOI: 10.1101/2020.03.13.991414
Posted 15 Mar 2020
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 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 critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-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 development of novel myeloid cellular therapies.
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