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

Proceedings of the National Academy of Sciences of the United States of America - Tập 112 Số 33 - Trang 10437-10442 - 2015
Kathrin Schumann1,2, Steven Lin3, Elizabeth W. Boyer1,2, Dimitre R. Simeonov4,1,2, Meena Subramaniam5,6,7, Rachel E. Gate5,6,7, Genevieve Haliburton1,2, Chun Ye6,7, Jeffrey A. Bluestone1, Jennifer A. Doudna8,3,9,10,11, Alexander Marson1,2,10
1Diabetes Center, University of California, San Francisco, CA 94143;
2Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
3Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
4Biomedical Sciences Graduate Program, University of California, San Francisco, CA 94143;
5Biological and Medical Informatics Graduate Program, University of California, San Francisco, CA 94158;
6Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences,
7Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143;
8Department of Chemistry, University of California, Berkeley, CA 94720; and
9Howard Hughes Medical Institute, University of California, Berkeley, CA 94720
10Innovative Genomics Initiative, University of California, Berkeley, CA 94720;
11Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Tóm tắt

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.

Từ khóa


Tài liệu tham khảo

JA Doudna, E Charpentier, Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

PD Hsu, ES Lander, F Zhang, Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

PK Mandal, , Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).

MV Maus, , Adoptive immunotherapy for cancer or viruses. Annu Rev Immunol 32, 189–225 (2014).

L Passerini, , CD4⁺ T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer. Sci Transl Med 5, 215ra174 (2013).

G Hütter, , Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med 360, 692–698 (2009).

CA Didigu, , Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood 123, 61–69 (2014).

P Tebas, , Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 370, 901–910 (2014).

NP Restifo, ME Dudley, SA Rosenberg, Adoptive immunotherapy for cancer: Harnessing the T cell response. Nat Rev Immunol 12, 269–281 (2012).

DL Porter, BL Levine, M Kalos, A Bagg, CH June, Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365, 725–733 (2011).

EK Moon, , Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 20, 4262–4273 (2014).

SL Topalian, CG Drake, DM Pardoll, Immune checkpoint blockade: A common denominator approach to cancer therapy. Cancer Cell 27, 450–461 (2015).

LB John, , Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells. Clin Cancer Res 19, 5636–5646 (2013).

P Genovese, , Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235–240 (2014).

S Kim, D Kim, SW Cho, J Kim, JS Kim, Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24, 1012–1019 (2014).

S Lin, BT Staahl, RK Alla, JA Doudna, Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

JA Zuris, , Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33, 73–80 (2015).

YH Sung, , Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 24, 125–131 (2014).

YR Zou, AH Kottmann, M Kuroda, I Taniuchi, DR Littman, Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599 (1998).

JF Berson, , A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains. J Virol 70, 6288–6295 (1996).

Y Feng, CC Broder, PE Kennedy, EA Berger, HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272, 872–877 (1996).

LS Symington, J Gautier, Double-strand break end resection and repair pathway choice. Annu Rev Genet 45, 247–271 (2011).

DY Guschin, , A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649, 247–256 (2010).

G Vahedi, , Helper T-cell identity and evolution of differential transcriptomes and epigenomes. Immunol Rev 252, 24–40 (2013).

KK Farh, , Genetic and epigenetic fine mapping of causal autoimmune disease variants. Nature 518, 337–343 (2015).

Thông tin
Thông tin xuất bản

Proceedings of the National Academy of Sciences of the United States of America

Tập 112 Số 33

10437-10442

Thông tin tác giả