Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

Contributed by Jennifer A. Doudna, June 29, 2015 (sent for review January 23, 2015)
July 27, 2015
112 (33) 10437-10442

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.

Abstract

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

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Data Availability

Data deposition: The sequence reported in this paper has been deposited in the National Center for Biotechnology Information (NCBI) BioSample database, www.ncbi.nlm.nih.gov/biosample/ (accession no. SAMN03850749).

Acknowledgments

We thank Mary Rieck, Jacqueline Howells, Amy Putnam, and Caroline Raffin in the J.A.B. laboratory; Richard Lao and the University of California at San Francisco (UCSF) Institute for Human Genetics Genomics Core; Michael Lee, Vinh Nguyen, and the UCSF Flow Cytometry Core; Amy Lee in the Cate laboratory; all members of the A.M., J.A.B., and J.A.D. laboratories for suggestions and technical assistance; and K. M. Ansel for critical reading of the manuscript. This research was supported by the UCSF Sandler Fellowship (to A.M.); a gift from Jake Aronov (to A.M.); NIH funding for the HIV Accessory & Regulatory Complexes Center (P50GM082250) (to A.M. and J.A.D.); a National MS Society Collaborative MS Research Center Award (to A.M. and J.A.D.); and the Howard Hughes Medical Institute (HHMI) (J.A.D.). S.L. is an HHMI Fellow of the Damon Runyon Cancer Research Foundation [DRG-(2176-13)], and G.E.H. is supported by an NIH training grant to UCSF Diabetes, Endocrinology and Metabolism (T32 DK741834).

Supporting Information

Supporting Information (PDF)
Supporting Information
pnas.1512503112.sd01.xlsx

References

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 112 | No. 33
August 18, 2015
PubMed: 26216948

Classifications

Data Availability

Data deposition: The sequence reported in this paper has been deposited in the National Center for Biotechnology Information (NCBI) BioSample database, www.ncbi.nlm.nih.gov/biosample/ (accession no. SAMN03850749).

Submission history

Published online: July 27, 2015
Published in issue: August 18, 2015

Keywords

  1. CRISPR/Cas9
  2. genome engineering
  3. Cas9 ribonucleoprotein
  4. RNP
  5. primary human T cells

Acknowledgments

We thank Mary Rieck, Jacqueline Howells, Amy Putnam, and Caroline Raffin in the J.A.B. laboratory; Richard Lao and the University of California at San Francisco (UCSF) Institute for Human Genetics Genomics Core; Michael Lee, Vinh Nguyen, and the UCSF Flow Cytometry Core; Amy Lee in the Cate laboratory; all members of the A.M., J.A.B., and J.A.D. laboratories for suggestions and technical assistance; and K. M. Ansel for critical reading of the manuscript. This research was supported by the UCSF Sandler Fellowship (to A.M.); a gift from Jake Aronov (to A.M.); NIH funding for the HIV Accessory & Regulatory Complexes Center (P50GM082250) (to A.M. and J.A.D.); a National MS Society Collaborative MS Research Center Award (to A.M. and J.A.D.); and the Howard Hughes Medical Institute (HHMI) (J.A.D.). S.L. is an HHMI Fellow of the Damon Runyon Cancer Research Foundation [DRG-(2176-13)], and G.E.H. is supported by an NIH training grant to UCSF Diabetes, Endocrinology and Metabolism (T32 DK741834).

Authors

Affiliations

Kathrin Schumann1
Diabetes Center, University of California, San Francisco, CA 94143;
Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
Steven Lin1
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
Eric Boyer
Diabetes Center, University of California, San Francisco, CA 94143;
Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
Dimitre R. Simeonov
Diabetes Center, University of California, San Francisco, CA 94143;
Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
Biomedical Sciences Graduate Program, University of California, San Francisco, CA 94143;
Meena Subramaniam
Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143;
Biological and Medical Informatics Graduate Program, University of California, San Francisco, CA 94158;
Rachel E. Gate
Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143;
Biological and Medical Informatics Graduate Program, University of California, San Francisco, CA 94158;
Genevieve E. Haliburton
Diabetes Center, University of California, San Francisco, CA 94143;
Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
Chun J. Ye
Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143;
Jeffrey A. Bluestone
Diabetes Center, University of California, San Francisco, CA 94143;
Jennifer A. Doudna2 [email protected]
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
Innovative Genomics Initiative, University of California, Berkeley, CA 94720;
Howard Hughes Medical Institute, University of California, Berkeley, CA 94720;
Department of Chemistry, University of California, Berkeley, CA 94720;
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
Alexander Marson2 [email protected]
Diabetes Center, University of California, San Francisco, CA 94143;
Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
Innovative Genomics Initiative, University of California, Berkeley, CA 94720;

Notes

2
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: K.S., S.L., J.A.D., and A.M. designed research; K.S., S.L., E.B., and D.R.S. performed research; K.S., S.L., E.B., D.R.S., M.S., R.E.G., G.E.H., C.J.Y., J.A.B., J.A.D., and A.M. analyzed data; and K.S., S.L., and A.M. wrote the paper.
1
K.S. and S.L. contributed equally to this work.

Competing Interests

Conflict of interest statement: J.A.D. is a co-founder of Caribou Biosciences Inc., Editas Medicine, and Intellia and is on the scientific advisory board of Caribou Biosciences Inc. The A.M. laboratory receives sponsored research support from Epinomics. A patent has been filed based on the findings described here.

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    Generation of knock-in primary human T cells using Cas9 ribonucleoproteins
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 33
    • pp. 10069-E4635

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