A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

Judd F Hultquist; Kathrin Schumann; Jonathan M Woo; Lara Manganaro; Michael J McGregor; Jennifer Doudna; Viviana Simon; Nevan J Krogan; Alexander Marson

doi:10.1016/j.celrep.2016.09.080

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

Cell Rep. 2016 Oct 25;17(5):1438-1452. doi: 10.1016/j.celrep.2016.09.080.

Authors

Judd F Hultquist¹, Kathrin Schumann², Jonathan M Woo³, Lara Manganaro⁴, Michael J McGregor¹, Jennifer Doudna⁵, Viviana Simon⁶, Nevan J Krogan⁷, Alexander Marson⁸

Affiliations

¹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA.
² Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁵ Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Lawrence Berkeley National Laboratory, Physical Biosciences Division, Berkeley, Berkeley, CA 94720, USA.
⁶ Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁷ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA. Electronic address: nevan.krogan@ucsf.edu.
⁸ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Divisions of Infectious Diseases and Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: alexander.marson@ucsf.edu.

Abstract

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4⁺ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

Keywords: CCR5; CRISPR/Cas9; CXCR4; HIV integrase; LEDGF; TNPO3; genome editing; host dependency factors; host-pathogen interactions; primary T cells.

Publication types

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

MeSH terms

CD4-Positive T-Lymphocytes / metabolism*
CD4-Positive T-Lymphocytes / virology*
CRISPR-Cas Systems / genetics*
Cells, Cultured
Gene Editing / methods*
Gene Knockout Techniques
HIV Infections / genetics*
HIV-1 / genetics
Host-Pathogen Interactions / genetics*
Humans
Intercellular Signaling Peptides and Proteins / metabolism
Receptors, CXCR4 / metabolism
Receptors, CXCR5 / metabolism
Reproducibility of Results
Ribonucleoproteins / metabolism*
beta Karyopherins / metabolism

Substances

CXCR4 protein, human
CXCR5 protein, human
Intercellular Signaling Peptides and Proteins
Receptors, CXCR4
Receptors, CXCR5
Ribonucleoproteins
TNPO3 protein, human
beta Karyopherins
lens epithelium-derived growth factor

Abstract

Publication types

MeSH terms

Substances

Grants and funding