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

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 withmore » 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.« less
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [3] ;  [4]
  1. Univ. of California, San Francisco, CA (United States)
  2. Univ. of California, Berkeley, CA (United States)
  3. Univ. of California, San Francisco, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  4. Univ. of California, San Francisco, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 33; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CRISPR/Cas9; genome engineering; Cas9 ribonucleoprotein; RNP primary; human T cells
OSTI Identifier:
1221821

Schumann, Kathrin, Lin, Steven, Boyer, Eric, Simeonov, Dimitre R., Subramaniam, Meena, Gate, Rachel E., Haliburton, Genevieve E., Ye, Chun J., Bluestone, Jeffrey A., Doudna, Jennifer A., and Marson, Alexander. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. United States: N. p., Web. doi:10.1073/pnas.1512503112.
Schumann, Kathrin, Lin, Steven, Boyer, Eric, Simeonov, Dimitre R., Subramaniam, Meena, Gate, Rachel E., Haliburton, Genevieve E., Ye, Chun J., Bluestone, Jeffrey A., Doudna, Jennifer A., & Marson, Alexander. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. United States. doi:10.1073/pnas.1512503112.
Schumann, Kathrin, Lin, Steven, Boyer, Eric, Simeonov, Dimitre R., Subramaniam, Meena, Gate, Rachel E., Haliburton, Genevieve E., Ye, Chun J., Bluestone, Jeffrey A., Doudna, Jennifer A., and Marson, Alexander. 2015. "Generation of knock-in primary human T cells using Cas9 ribonucleoproteins". United States. doi:10.1073/pnas.1512503112. https://www.osti.gov/servlets/purl/1221821.
@article{osti_1221821,
title = {Generation of knock-in primary human T cells using Cas9 ribonucleoproteins},
author = {Schumann, Kathrin and Lin, Steven and Boyer, Eric and Simeonov, Dimitre R. and Subramaniam, Meena and Gate, Rachel E. and Haliburton, Genevieve E. and Ye, Chun J. and Bluestone, Jeffrey A. and Doudna, Jennifer A. and Marson, Alexander},
abstractNote = {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.},
doi = {10.1073/pnas.1512503112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 33,
volume = 112,
place = {United States},
year = {2015},
month = {7}
}