@article{74cb31de1ae048f0bec28c99308659c7,
title = "A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells",
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",
author = "Hultquist, {Judd F.} and Kathrin Schumann and Woo, {Jonathan M.} and Lara Manganaro and McGregor, {Michael J.} and Jennifer Doudna and Viviana Simon and Krogan, {Nevan J.} and Alexander Marson",
note = "Funding Information: We thank all members of A.M., N.J.K., and V.S. labs for suggestions and technical assistance. This research was supported by the UCSF MPHD T32 Training Grant (J.F.H.), a fellowship of the Deutsche Forschungsgemeinschaft (SCHU 3020/2-1, K.S.), a UCSF Sandler Fellowship (A.M.), a gift from Jake Aronov (A.M.), the NIH/NIDA Avenir New Innovator Award (DP2DA042423, A.M.), NIH/NIAID funding for HIV studies (R01 AI064001, R01 AI125173, and R01 AI120998, V.S.), NIH/NIGMS funding for the HIV Accessory and Regulatory Complexes (HARC) Center (P50 GM082250, A.M. and N.J.K.), NIH funding for the FluOMICs cooperative agreement (U19 AI106754, J.F.H. and N.J.K.), NIH/NIAID funding for the HIV Immune Networks Team (P01 AI090935, N.J.K. and V.S.), and NIH funding for the UCSF-Gladstone Institute of Virology and Immunology Center for AIDS Research (CFAR; P30 AI027763). Special thanks to Ethan Brookes, Matthew Hall, and Olivier Cantada at Lonza Bioscience for their support with the nucleofection transfection technology. We also thank Anja Smith and Darrick Chow at Dharmacon for support and assistance with crRNA and tracrRNA synthesis. A patent has been filed on the use of Cas9 RNPs to edit the genome of human primary cells (A.M., J.A.D., and K.S.). A.M. serves as an advisor to Juno Therapeutics, and the A.M. lab has sponsored research agreements with Juno Therapeutics and Epinomics. J.D. is a co-founder of Editas Medicine, Intellia Therapeutics, and Caribou Biosciences and serves as a scientific advisor to Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, and Driver. Publisher Copyright: {\textcopyright} 2016 The Author(s)",
year = "2016",
month = oct,
day = "25",
doi = "10.1016/j.celrep.2016.09.080",
language = "English (US)",
volume = "17",
pages = "1438--1452",
journal = "Cell Reports",
issn = "2211-1247",
publisher = "Cell Press",
number = "5",
}
