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Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Authors :
David E. Gordon
Weihao Zheng
Satish K. Pillai
Anke Meyer-Franke
Alexander Marson
Mohamed S. Bouzidi
Krystal A. Fontaine
Joseph Hiatt
Jonathan M. Budzik
Joel D. Ernst
Jason A. Wojcechowskyj
Ujjwal Rathore
Nevan J. Krogan
Kelsey M. Haas
Judd F. Hultquist
Theodore L. Roth
Jeffery S. Cox
Devin A. Cavero
Michael J. McGregor
Publication Year :
2020
Publisher :
Cold Spring Harbor Laboratory, 2020.

Abstract

SummaryGenome 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.

Details

Database :
OpenAIRE
Accession number :
edsair.doi.dedup.....8290ee4feeb36c7da93a729b5cf03042