期刊
NATURE BIOMEDICAL ENGINEERING
卷 1, 期 11, 页码 878-888出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41551-017-0145-2
关键词
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资金
- National Institutes of Health [R01AI020047, R01AI077595, RO1HL107630, HL107440, UC4DK104218, U19HL129903]
- Translational Research Program (Boston Children's Hospital)
- Pedals for Pediatrics (Dana-Farber Cancer Institute)
- Leona M. and Harry B. Helmsley Charitable Trust
- New York Stem Cell Foundation
Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.
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