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
Affiliations
Judd F. Hultquist
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
Kathrin Schumann
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
Jonathan M. Woo
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
Lara Manganaro
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
Michael J. McGregor
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
Jennifer Doudna
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
Viviana Simon
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
Nevan J. Krogan
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; Corresponding author
Alexander Marson
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; Corresponding author
Summary: 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. : Hultquist et al. report a high-throughput platform for the efficient, multiplex editing of host factors that control HIV infection in primary CD4+ T cells. Arrayed electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs) permits the rapid generation of isogenic human cells with ablated candidate factors and identifies gene modifications that provide viral resistance. Keywords: CRISPR/Cas9, HIV integrase, primary T cells, genome editing, CXCR4, CCR5, LEDGF, TNPO3, host-pathogen interactions, host dependency factors
