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Nat Protoc. 2019 Jan;14(1):1-27. doi: 10.1038/s41596-018-0069-7.

CRISPR-Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV-host factor interactions.

Hultquist JF1,2,3,4, Hiatt J3,5,6,7,8, Schumann K5,6, McGregor MJ1,2,3, Roth TL5,6,7,8, Haas P1,2,3, Doudna JA9,10,11,12,13, Marson A14,15,16,17,18,19, Krogan NJ20,21,22.

Author information

1
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
2
Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA.
3
Institute for Virology and Immunology, J. David Gladstone Institutes, San Francisco, CA, USA.
4
Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
5
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
6
Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
7
Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
8
Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
9
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
10
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
11
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
12
Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
13
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
14
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
15
Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
16
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. alexander.marson@ucsf.edu.
17
Department of Medicine, University of California, San Francisco, CA, USA. alexander.marson@ucsf.edu.
18
UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. alexander.marson@ucsf.edu.
19
Chan Zuckerberg Biohub, San Francisco, CA, USA. alexander.marson@ucsf.edu.
20
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. nevan.krogan@ucsf.edu.
21
Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA. nevan.krogan@ucsf.edu.
22
Institute for Virology and Immunology, J. David Gladstone Institutes, San Francisco, CA, USA. nevan.krogan@ucsf.edu.

Abstract

CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4+ T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4+ T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4+ T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.

PMID:
30559373
DOI:
10.1038/s41596-018-0069-7

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