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

2015; National Academy of Sciences; Volume: 112; Issue: 33 Linguagem: Inglês

10.1073/pnas.1512503112

1091-6490

Kathrin Schumann, Steven Lin, Eric Boyer, Dimitre R. Simeonov, Meena Subramaniam, Rachel E. Gate, Genevieve Haliburton, Chun Ye, Jeffrey A. Bluestone, Jennifer A. Doudna, Alexander Marson,

RNA Interference and Gene Delivery

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.