Generation of knock-in primary human T cells using Cas9 ribonucleoproteins
Open accessAbstract
Significance: T-cell genome engineering holds great promise for cancer immunotherapies and cell-based therapies for HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been inefficient. We achieved efficient genome editing by delivering Cas9 protein pre-assembled with guide RNAs. These active Cas9 ribonucleoproteins (RNPs) enabled successful Cas9-mediated homology-directed repair in primary human T cells. Cas9 RNPs provide a programmable tool to replace specific nucleotide sequences in the genome of mature immune cells—a longstanding goal in the field. These studies establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.
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It 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 [18]
The Discussion (page unavailable) describes fast editing, rapid Cas9 turnover and efficient editing in human T cells. The Results (page unavailable) found rare indels at two predicted off-target sites at rates comparable to controls. But the paper does not establish that RNP delivery minimizes off-target effects compared with other delivery methods; the Discussion says further testing is needed. (LLM, conf=0.86)
Experimental — accuracy not yet measuredThe 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 [44]
The Results and Methods describe electroporating preassembled Cas9 protein–guide RNA complexes into primary human T cells, and the Discussion describes advantages of this approach over other delivery methods. The paper does not establish that electroporation of Cas9 RNPs is the currently favored ex vivo delivery method across primary cells generally. (LLM, conf=0.84)
Experimental — accuracy not yet measuredElectroporation 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 Schumann et al., 2015
Effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult due to the large size of Cas9 protein (~160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA.
“However, due to the large size of Cas9 protein (~ 160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA, the effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult [88–91].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [91]
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“We note that the T7E1 assay often underestimates the rate of indel formation, potentially due to self-hybridization of the alleles that carry the indel, incomplete duplex melting, and inefficient cleavage of single nucleotide indels (Schumann et al., 2015).”
BackgroundMethodA compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing
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- Authors
- Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., Haliburton, G. E., Ye, C. J., Bluestone, J. A., Doudna, J. A., & Marson, A.
- Affiliations
- University of California, San Francisco (US)
- University of California, Berkeley (US)
- Howard Hughes Medical Institute (US)
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2015-07-27
- Volume
- 112
- Issue
- 33
- Pages
- 10437-10442
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC4547290
- OpenAlex
- W2164934988
- Semantic Scholar
- 8ff28a21bb88c381f81d9cb63b5c7009c5251447
- Cited by
- 702
- References
- 25
- Citations by year
- 2026: 31 · 2025: 33 · 2024: 45 · 2023: 54 · 2022: 74 · 2021: 77 · 2020: 75 · 2019: 72 · 2018: 93 · 2017: 70 · 2016: 67 · 2015: 10
- FWCI
- 34.70
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- Damon Runyon Cancer Research Foundation
- National Institutes of Health
- University of California, San Francisco
- National Institute of General Medical Sciences (T32GM008284, P50GM082250, T32GM067547, T32GM008568)
- National Institute of Diabetes and Digestive and Kidney Diseases (T32 DK741834, P30DK063720)
- National Institute of Biomedical Imaging and Bioengineering (T32EB009383)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- CRISPR/Cas9, Cas9 ribonucleoprotein, CD4+ T cells, CXCR4, PD-1, homology-directed repair, electroporation, T cell exhaustion, cell surface expression
- MeSH terms
- Bacterial Proteins, Cell Line, Endonucleases, Genetic Engineering, Humans, Leukocytes, Mononuclear, Ribonucleoproteins, T-Lymphocytes, CD4-Positive T-Lymphocytes, Genome, Electroporation, Receptors, CXCR4, Gene Knock-In Techniques, High-Throughput Nucleotide Sequencing, Clustered Regularly Interspaced Short Palindromic Repeats
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