skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

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 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:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1221821
Grant/Contract Number:  
AC02-05CH11231
Resource 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)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CRISPR/Cas9; genome engineering; Cas9 ribonucleoprotein; RNP primary; human T cells

Citation Formats

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., 2015. 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. Mon . "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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 125 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

The new frontier of genome engineering with CRISPR-Cas9
journal, November 2014


Development and Applications of CRISPR-Cas9 for Genome Engineering
journal, June 2014


Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR/Cas9
journal, November 2014


Adoptive Immunotherapy for Cancer or Viruses
journal, March 2014


CD4+ T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer
journal, December 2013


Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation
journal, February 2009

  • Hütter, Gero; Nowak, Daniel; Mossner, Maximilian
  • New England Journal of Medicine, Vol. 360, Issue 7
  • DOI: 10.1056/NEJMoa0802905

Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection
journal, January 2014


Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV
journal, March 2014

  • Tebas, Pablo; Stein, David; Tang, Winson W.
  • New England Journal of Medicine, Vol. 370, Issue 10
  • DOI: 10.1056/NEJMoa1300662

Adoptive immunotherapy for cancer: harnessing the T cell response
journal, March 2012

  • Restifo, Nicholas P.; Dudley, Mark E.; Rosenberg, Steven A.
  • Nature Reviews Immunology, Vol. 12, Issue 4
  • DOI: 10.1038/nri3191

Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia
journal, August 2011

  • Porter, David L.; Levine, Bruce L.; Kalos, Michael
  • New England Journal of Medicine, Vol. 365, Issue 8
  • DOI: 10.1056/NEJMoa1103849

Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy
journal, April 2015


Anti-PD-1 Antibody Therapy Potently Enhances the Eradication of Established Tumors By Gene-Modified T Cells
journal, July 2013


Targeted genome editing in human repopulating haematopoietic stem cells
journal, May 2014

  • Genovese, Pietro; Schiroli, Giulia; Escobar, Giulia
  • Nature, Vol. 510, Issue 7504
  • DOI: 10.1038/nature13420

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins
journal, April 2014


Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery
journal, December 2014


Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo
journal, October 2014

  • Zuris, John A.; Thompson, David B.; Shu, Yilai
  • Nature Biotechnology, Vol. 33, Issue 1
  • DOI: 10.1038/nbt.3081

Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases
journal, November 2013


Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development
journal, June 1998

  • Zou, Yong-Rui; Kottmann, Andreas H.; Kuroda, Masahiko
  • Nature, Vol. 393, Issue 6685
  • DOI: 10.1038/31269

HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor
journal, May 1996


Double-Strand Break End Resection and Repair Pathway Choice
journal, December 2011


Helper T-cell identity and evolution of differential transcriptomes and epigenomes
journal, February 2013

  • Vahedi, Golnaz; C. Poholek, Amanda; Hand, Timothy W.
  • Immunological Reviews, Vol. 252, Issue 1
  • DOI: 10.1111/imr.12037

Genetic and epigenetic fine mapping of causal autoimmune disease variants
journal, October 2014

  • Farh, Kyle Kai-How; Marson, Alexander; Zhu, Jiang
  • Nature, Vol. 518, Issue 7539
  • DOI: 10.1038/nature13835

    Works referencing / citing this record:

    Primary allogeneic mitochondrial mix (PAMM) transfer/transplant by MitoCeption to address damage in PBMCs caused by ultraviolet radiation
    journal, June 2019


    Spontaneous HTLV-1 transcription is accompanied by distinct epigenetic changes in the 5′ and 3′ long terminal repeats
    journal, January 2018


    Metabolic reprogramming of human CD8 + memory T cells through loss of SIRT1
    journal, November 2017

    • Jeng, Mark Y.; Hull, Philip A.; Fei, Mingjian
    • The Journal of Experimental Medicine, Vol. 215, Issue 1
    • DOI: 10.1084/jem.20161066

    Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders
    journal, January 2016


    Strategies for nonviral nanoparticle‐based delivery of CRISPR/Cas9 therapeutics
    journal, December 2019

    • Chen, Fengqian; Alphonse, Martin; Liu, Qi
    • WIREs Nanomedicine and Nanobiotechnology
    • DOI: 10.1002/wnan.1609

    Humanized model mice by genome editing and engraftment technologies
    journal, June 2018


    Genetic editing and interrogation with Cpf1 and caged truncated pre-tRNA-like crRNA in mammalian cells
    journal, July 2018


    Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells
    journal, October 2018


    Recent advances in DNA-free editing and precise base editing in plants
    journal, November 2017

    • Zhang, Yi; Gao, Caixia
    • Emerging Topics in Life Sciences, Vol. 1, Issue 2
    • DOI: 10.1042/etls20170021