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Proc Natl Acad Sci U S A. 2017 Dec 12;114(50):E10745-E10754. doi: 10.1073/pnas.1711979114. Epub 2017 Nov 28.

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks.

Author information

1
Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore MD 21205 apaix1@jhmi.edu gseydoux@jhmi.edu.
2
Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore MD 21205.

Abstract

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

KEYWORDS:

CRISPR; HDR; PCR repair template; SDSA; short homology arms

PMID:
29183983
PMCID:
PMC5740635
DOI:
10.1073/pnas.1711979114
[Indexed for MEDLINE]
Free PMC Article

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