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Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10437-42. doi: 10.1073/pnas.1512503112. Epub 2015 Jul 27.

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

Author information

1
Diabetes Center, University of California, San Francisco, CA 94143; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
2
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
3
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;
4
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;
5
Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143;
6
Diabetes Center, University of California, San Francisco, CA 94143;
7
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.marson@ucsf.edu doudna@berkeley.edu.
8
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; alexander.marson@ucsf.edu doudna@berkeley.edu.

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.

KEYWORDS:

CRISPR/Cas9; Cas9 ribonucleoprotein; RNP; genome engineering; primary human T cells

PMID:
26216948
PMCID:
PMC4547290
DOI:
10.1073/pnas.1512503112
[Indexed for MEDLINE]
Free PMC Article

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