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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We thank L. Pache, E. Battivelli, and members of the Marson and Krogan labs for critical feedback and testing of the protocol. This research was supported by amfAR grant 109504-61-RKRL, using funds raised by generationCURE (J.F.H.); a fellowship of the Deutsche Forschungsgemeinschaft (SCHU3020/2-1; K.S.); the UCSF Sandler Fellowship (A.M.), a gift from Jake Aronov (A.M.); NIH/NIGMS funding for the HIV Accessory & Regulatory Complexes (HARC) Center (P50 GM082250; A.M., J.D., 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.); NIH funding for the Dengue Human Immunology Project Consortium (DHIPC, U19 AI118610; N.J.K.); NIH funding for the study of innate immune responses to intracellular pathogens (R01 AI120694 and P01 AI063302; N.J.K.); NIH funding for the UCSF-Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR, P30 AI027763); and an NIH/NIDA grant (DP2 DA042423-01; A.M.). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, is a Chan Zuckerberg Biohub Investigator, and has received funding from the Innovative Genomics Institute (IGI). Special thanks to E. Brookes, M. Hall, and O. Cantada at Lonza Bioscience for their support in regard to the nucleofection transfection technology; D. Chow at Dharmacon for his support in regard to gRNA synthesis; T. Brown at Thermo Fisher Scientific for his support in regard to the Attune NxT Flow Cytometer, and C. Jeans at the University of California, Berkeley Macrolab for the production of Cas9 protein.
An intellectual property patent application has been filed for the use of CRISPR–Cas9 RNPs to edit the genome of human primary hematopoietic cells. A.M. is a cofounder of Spotlight Therapeutics, serves on the scientific advisory board of PACT Pharma, and was previously an adviser to Juno Therapeutics. The Marson lab has received sponsored research funding from Juno Therapeutics, Epinomics, and Sanofi, and a gift from Gilead. The remaining authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Schumann, K. et al. Proc. Natl. Acad. Sci. USA 112, 10437–10442 (2015): https://doi.org/10.1073/pnas.1512503112
Hultquist, J. F. et al. Cell Rep. 17, 1438–1452 (2016): https://doi.org/10.1016/j.celrep.2016.09.080
Park, R. J. et al. Nat. Genet. 49, 193–203 (2017): https://doi.org/10.1038/ng.3741
Integrated supplementary information
Supplementary Figure 1 Gating strategy for flow cytometry analysis of immunostained and infected primary CD4+ T cells.
(a) Flow cytometry contour plot (left) and histogram (right) depicting cell size and CD4 levels on the cell surface of isolated PBMCs (Steps 32 and 47). A live cell gate (‘PBMCs’) is first applied to a forward versus side scatter contour plot before analysis of CD4 levels by histogram. Samples were run on an Attune NxT Flow cytometer and analyzed using FlowJo software v10.1 (n >100,000 events). A similar strategy is used for the analysis of CD4, CD25, or CXCR4 levels in various cell populations before and after isolation, stimulation, and editing. (b) Flow cytometry dot plots depicting cell size (left) and GFP levels (right) of infected primary CD4+ T cells (Step 108). A live cell gate (‘Primary T Cells’) is first applied to a forward versus side scatter dot plot before analysis of GFP levels. An uninfected, GFP-negative control is first run to define a threshold for GFP gating. Samples were run on an Attune NxT Flow cytometer and analyzed using FlowJo software v10.1 (n >10,000 events).
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Hultquist, J.F., Hiatt, J., Schumann, K. et al. CRISPR–Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV–host factor interactions. Nat Protoc 14, 1–27 (2019). https://doi.org/10.1038/s41596-018-0069-7
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