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CRISPR–Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV–host factor interactions


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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Fig. 1: Overview of primary T-cell editing using CRISPR–Cas9 RNPs. Primary CD4+ T cells are isolated from donor blood and activated.
Fig. 2: Results from primary T-cell isolation and editing.
Fig. 3: Overview of HIV-1 virus preparation.
Fig. 4: HIV spreading infection results over a time course of infection in primary CD4+ T cells.


  1. 1.

    Harris, R. S., Hultquist, J. F. & Evans, D. T. The restriction factors of human immunodeficiency virus. J. Biol. Chem. 287, 40875–40883 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Goff, S. P. Host factors exploited by retroviruses. Nat. Rev. Microbiol. 5, 253–263 (2007).

    CAS  Article  Google Scholar 

  3. 3.

    Hsu, T. H. & Spindler, K. R. Identifying host factors that regulate viral infection. PLoS Pathog. 8, e1002772 (2012).

    CAS  Article  Google Scholar 

  4. 4.

    Dorr, P. et al. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob. Agents Chemother. 49, 4721–4732 (2005).

    CAS  Article  Google Scholar 

  5. 5.

    Gulick, R. M. et al. Maraviroc for previously treated patients with R5 HIV-1 infection. N. Engl. J. Med. 359, 1429–1441 (2008).

    CAS  Article  Google Scholar 

  6. 6.

    Bushman, F. D. et al. Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog. 5, e1000437 (2009).

    Article  Google Scholar 

  7. 7.

    Yeung, M. L., Houzet, L., Yedavalli, V. S. & Jeang, K. T. A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication. J. Biol. Chem. 284, 19463–19473 (2009).

    CAS  Article  Google Scholar 

  8. 8.

    Park, R. J. et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat. Genet. 49, 193–203 (2017).

    CAS  Article  Google Scholar 

  9. 9.

    Pan, C., Kumar, C., Bohl, S., Klingmueller, U. & Mann, M. Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol. Cell. Proteomics 8, 443–450 (2009).

    CAS  Article  Google Scholar 

  10. 10.

    Alge, C. S., Hauck, S. M., Priglinger, S. G., Kampik, A. & Ueffing, M. Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival. J. Proteome Res. 5, 862–878 (2006).

    CAS  Article  Google Scholar 

  11. 11.

    Lorsch, J. R., Collins, F. S. & Lippincott-Schwartz, J. Cell biology. Fixing problems with cell lines. Science 346, 1452–1453 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Hughes, P., Marshall, D., Reid, Y., Parkes, H. & Gelber, C. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Biotechniques 43, 575 (2007).

    CAS  Article  Google Scholar 

  13. 13.

    American Type Culture Collection Standards Development Organization Workgroup ASN-0002. Cell line misidentification: the beginning of the end. Nat. Rev. Cancer 10, 441–448 (2010).

    Article  Google Scholar 

  14. 14.

    Hultquist, J. F. et al. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV-host interactions in primary human T cells. Cell Rep. 17, 1438–1452 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 10437–10442 (2015).

    CAS  Article  Google Scholar 

  16. 16.

    Brass, A. L. et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science 319, 921–926 (2008).

    CAS  Article  Google Scholar 

  17. 17.

    Konig, R. et al. Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 135, 49–60 (2008).

    CAS  Article  Google Scholar 

  18. 18.

    Zhou, H. et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe 4, 495–504 (2008).

    CAS  Article  Google Scholar 

  19. 19.

    Pache, L., Konig, R. & Chanda, S. K. Identifying HIV-1 host cell factors by genome-scale RNAi screening. Methods 53, 3–12 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Llano, M. et al. An essential role for LEDGF/p75 in HIV integration. Science 314, 461–464 (2006).

    CAS  Article  Google Scholar 

  21. 21.

    Llano, M. et al. LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes. J. Virol. 78, 9524–9537 (2004).

    CAS  Article  Google Scholar 

  22. 22.

    Fadel, H. J. et al. TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism. J. Virol. 88, 9704–9717 (2014).

    Article  Google Scholar 

  23. 23.

    Shun, M. C. et al. LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev. 21, 1767–1778 (2007).

    CAS  Article  Google Scholar 

  24. 24.

    Jackson, A. L. & Linsley, P. S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat. Rev. Drug Discov. 9, 57–67 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Jackson, A. L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187 (2006).

    CAS  Article  Google Scholar 

  26. 26.

    Tripathi, S. et al. Meta- and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding. Cell Host Microbe 18, 723–735 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Pichlmair, A. et al. Viral immune modulators perturb the human molecular network by common and unique strategies. Nature 487, 486–490 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009).

    CAS  Article  Google Scholar 

  29. 29.

    Deans, R. M. et al. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification. Nat. Chem. Biol. 12, 361–366 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Heaton, N. S. et al. Targeting viral proteostasis limits influenza virus, HIV, and dengue virus infection. Immunity 44, 46–58 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Lin, S., Staahl, B., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3, e04766 (2014).

  32. 32.

    Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  Article  Google Scholar 

  34. 34.

    Mandal, P. K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016).

    Article  Google Scholar 

  36. 36.

    Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  Article  Google Scholar 

  37. 37.

    Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    CAS  Article  Google Scholar 

  39. 39.

    Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Fu, B. X., Hansen, L. L., Artiles, K. L., Nonet, M. L. & Fire, A. Z. Landscape of target:guide homology effects on Cas9-mediated cleavage. Nucleic Acids Res. 42, 13778–13787 (2014).

    CAS  Article  Google Scholar 

  41. 41.

    Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife 5, e19760 (2016).

  42. 42.

    Horlbeck, M. A. et al. Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife 5, e12677 (2016).

  43. 43.

    Isaac, R. S. et al. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function. Elife 5, e13450 (2016).

  44. 44.

    Larson, M. H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

    CAS  Article  Google Scholar 

  45. 45.

    Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    CAS  Article  Google Scholar 

  46. 46.

    Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    CAS  Article  Google Scholar 

  47. 47.

    Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    CAS  Article  Google Scholar 

  48. 48.

    Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).

    CAS  Article  Google Scholar 

  49. 49.

    Heigwer, F., Kerr, G. & Boutros, M. E-CRISP: fast CRISPR target site identification. Nat. Methods 11, 122–123 (2014).

    CAS  Article  Google Scholar 

  50. 50.

    Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

    Article  Google Scholar 

  51. 51.

    Haeussler, M. & Concordet, J. P. Genome editing with CRISPR-Cas9: can it get any better? J. Genetics Genomics 43, 239–250 (2016).

    Article  Google Scholar 

  52. 52.

    Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

    CAS  Article  Google Scholar 

  53. 53.

    Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018).

    CAS  Article  Google Scholar 

  54. 54.

    Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    CAS  Article  Google Scholar 

  55. 55.

    Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).

    CAS  Article  Google Scholar 

  56. 56.

    Blomen, V. A. et al. Gene essentiality and synthetic lethality in haploid human cells. Science 350, 1092–1096 (2015).

    CAS  Article  Google Scholar 

  57. 57.

    Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).

    CAS  Article  Google Scholar 

  58. 58.

    Healey, D. S., Jowett, J. B., Beaton, F., Maskill, W. J. & Gust, I. D. Comparison of enzyme immunoassay and reverse transcriptase assay for detection of HIV in culture supernates. J. Virol. Methods 17, 237–245 (1987).

    CAS  Article  Google Scholar 

  59. 59.

    Healey, D. S., Maskill, W. J., Neate, E. V., Beaton, F. & Gust, I. D. A preliminary evaluation of five HIV antigen detection assays. J. Virol. Methods 20, 115–125 (1988).

    CAS  Article  Google Scholar 

  60. 60.

    Gupta, P., Balachandran, R., Grovit, K., Webster, D. & Rinaldo, C. Jr. Detection of human immunodeficiency virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay. J. Clin. Microbiol. 25, 1122–1125 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Munch, J. et al. Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses. J. Virol. 81, 13852–13864 (2007).

    Article  Google Scholar 

  62. 62.

    O’Doherty, U., Swiggard, W. J. & Malim, M. H. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J. Virol. 74, 10074–10080 (2000).

    Article  Google Scholar 

  63. 63.

    Mandegar, M. A. et al. CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18, 541–553 (2016).

    CAS  Article  Google Scholar 

  64. 64.

    Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    Article  Google Scholar 

  65. 65.

    Koressaar, T. & Remm, M. Enhancements and modifications of primer design program Primer3. Bioinformatics 23, 1289–1291 (2007).

    CAS  Article  Google Scholar 

  66. 66.

    Untergasser, A. et al. Primer3--new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).

    CAS  Article  Google Scholar 

  67. 67.

    Wehrly, K. & Chesebro, B. p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods 12, 288–293 (1997).

    CAS  Article  Google Scholar 

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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.

Author information




J.F.H., J.H., K.S., N.J.K., and A.M. designed the experimental procedure, which benefited from additional input from J.H., T.L.R., M.J.M., P.H., and J.D. Quality control and infection data were collected by J.F.H., J.H., and M.J.M. The figures were designed and assembled by J.F.H. with input from J.H. The text was written by J.F.H., J.H., A.M., and N.J.K., with critical input from M.J.M., P.H., K.S., T.L.R., and J.D.

Corresponding authors

Correspondence to Alexander Marson or Nevan J. Krogan.

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Competing interests

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.

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Key references using this protocol

Schumann, K. et al. Proc. Natl. Acad. Sci. USA 112, 10437–10442 (2015):

Hultquist, J. F. et al. Cell Rep. 17, 1438–1452 (2016):

Park, R. J. et al. Nat. Genet. 49, 193–203 (2017):

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).

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