Journal
NATURE PROTOCOLS
Volume 14, Issue 1, Pages 1-27Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-018-0069-7
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Funding
- amfAR [109504-61-RKRL]
- generationCURE
- Deutsche Forschungsgemeinschaft [SCHU3020/2-1]
- UCSF Sandler Fellowship
- NIH/NIGMS funding for the HIV Accessory & Regulatory Complexes (HARC) Center [P50 GM082250]
- NIH/NIAID funding for the HIV Immune Networks Team [P01 AI090935]
- NIH funding for the Dengue Human Immunology Project Consortium (DHIPC) [U19 AI118610]
- NIH funding for the study of innate immune responses to intracellular pathogens [R01 AI120694, P01 AI063302]
- NIH funding for the UCSF-Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR) [P30 AI027763]
- NIH/NIDA grant [DP2 DA042423-01]
- Burroughs Wellcome Fund
- Innovative Genomics Institute (IGI)
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI120694, P30AI027763, U19AI118610, K22AI136691, P01AI063302, U19AI106754] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [T32DK007418, F30DK120213] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P50GM082250, T32GM007618] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON DRUG ABUSE [DP2DA042423] Funding Source: NIH RePORTER
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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.
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