A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells
Open accessAbstract
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.
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The currently favored form of ex vivo delivery to primary cells is electroporation of Cas9 as a preformed protein-RNA (RNP) complex.
“The currently favored form of ex vivo delivery to primary cells is electroporation of Cas9 as a preformed protein-RNA (ribonucleoprotein, RNP) complex 44,56 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [56]
The Results and Experimental Procedures describe assembling Cas9 protein with RNA before electroporating the complex into primary human CD4+ T cells. The Introduction discusses advantages of this approach, but the paper does not establish that it is the currently favored delivery method across primary cells generally. (LLM, conf=0.86)
Experimental — accuracy not yet measuredIt reports that ribonucleoprotein delivery gives a brief pulse of editing machinery, producing efficient editing with fewer off-target effects.
“RNP delivery results in an initially high level of the genome editing machinery followed by rapid decay, leading to a “fast on, fast off” strategy that results in highly efficient editing while minimizing OTEs13–19.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [17]
Electroporation of Cas9 protein pre-loaded with sgRNAs efficiently edits primary human T cells.
“We previously showed efficient gene editing of primary human T cells by electroporation of Cas9 protein pre-loaded in vitro with sgRNAs (Hultquist et al., 2016; Schumann et al., 2015).”
From Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function (2018) · cites this paper as Hultquist et al., 2016
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References 88 · Cited by 170
- Authors
- Hultquist, J. F., Schumann, K., Woo, J. M., Manganaro, L., McGregor, M. J., Doudna, J., Simon, V., Krogan, N. J., & Marson, A.
- Affiliations
- QB3 (US)
- University of California, San Francisco (US)
- Icahn School of Medicine at Mount Sinai (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Krogan, Nevan J.
- Marson, Alexander
- ORCID
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2016-10
- Volume
- 17
- Issue
- 5
- Pages
- 1438-1452
- ISSN
- 2211-1247 (ISSN-L)
- PMC
- PMC5123761
- OpenAlex
- W2536383328
- Semantic Scholar
- 002ca904d8233b745e54deefc776b9cacd290ccb
- Cited by
- 170
- References
- 88
- Citations by year
- 2026: 5 · 2025: 8 · 2024: 7 · 2023: 22 · 2022: 22 · 2021: 17 · 2020: 18 · 2019: 20 · 2018: 33 · 2017: 16 · 2016: 2
- FWCI
- 12.30
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Reports (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
- Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- KOPS (University of Konstanz) (submittedVersion, cc-by-nc-nd)
- Funders
- Houston Advanced Research Center
- Deutsche Forschungsgemeinschaft
- National Institutes of Health (AI027763, GM082250)
- Center for AIDS Research, University of Washington (P30-AI027763)
- University of California, San Francisco (U19 AI106754, P30AI027763, GM082250)
- Juno Therapeutics
- National Institute on Drug Abuse (DP2DA042423)
- National Institute of General Medical Sciences (P50GM082250, GM082250)
- National Institute of Allergy and Infectious Diseases (AI120998, AI 090935, AI027763, P01AI090935, R01AI120998, AI064001, R01AI064001, AI106754, U19AI106754, T32AI060537, P30AI027763)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- CRISPR/Cas9 ribonucleoprotein, CXCR4, CCR5, LEDGF, TNPO3, HIV tropism, HIV integrase, host restriction factors, HIV cure
- MeSH terms
- Gene Editing, Cells, Cultured, Humans, Ribonucleoproteins, Reproducibility of Results, CD4-Positive T-Lymphocytes, HIV-1, HIV Infections, Receptors, CXCR4, beta Karyopherins, Intercellular Signaling Peptides and Proteins, Receptors, CXCR5, Host-Pathogen Interactions, Gene Knockout Techniques, CRISPR-Cas Systems
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