Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

Joseph Hiatt; Devin A Cavero; Michael J McGregor; Weihao Zheng; Jonathan M Budzik; Theodore L Roth; Kelsey M Haas; David Wu; Ujjwal Rathore; Anke Meyer-Franke; Mohamed S Bouzidi; Eric Shifrut; Youjin Lee; Vigneshwari Easwar Kumar; Eric V Dang; David E Gordon; Jason A Wojcechowskyj; Judd F Hultquist; Krystal A Fontaine; Satish K Pillai; Jeffery S Cox; Joel D Ernst; Nevan J Krogan; Alexander Marson

doi:10.1016/j.celrep.2021.109105

Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

Cell Rep. 2021 May 11;35(6):109105. doi: 10.1016/j.celrep.2021.109105.

Authors

Joseph Hiatt¹, Devin A Cavero², Michael J McGregor³, Weihao Zheng⁴, Jonathan M Budzik⁵, Theodore L Roth¹, Kelsey M Haas³, David Wu⁶, Ujjwal Rathore², Anke Meyer-Franke⁷, Mohamed S Bouzidi⁸, Eric Shifrut², Youjin Lee², Vigneshwari Easwar Kumar², Eric V Dang⁹, David E Gordon³, Jason A Wojcechowskyj³, Judd F Hultquist¹⁰, Krystal A Fontaine⁷, Satish K Pillai⁸, Jeffery S Cox¹¹, Joel D Ernst⁴, Nevan J Krogan¹², Alexander Marson¹³

Affiliations

¹ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
² J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
³ J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA.
⁴ Department of Medicine, Division of Experimental Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ J. David Gladstone Institutes, San Francisco, CA 94158, USA.
⁸ Vitalant Research Institute, San Francisco, CA 94118, USA; Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁹ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; Division of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
¹¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
¹² J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: [email protected].
¹³ J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94129, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address: [email protected].

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.

Keywords: CRISPR; Cas9; dendritic cells; electroporation; host-pathogen interactions; knockout; macrophages; monocytes; myeloid cells; ribonculeoproteins (RNPs).

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems / genetics*
Genome / genetics*
Humans
Mice
Myeloid Cells / metabolism*
Ribonucleoproteins / metabolism*

Substances

Ribonucleoproteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding