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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems
  • DNA Breaks, Double-Stranded
  • DNA Repair*
  • Endonucleases / metabolism*
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Mice
  • Polymerase Chain Reaction
  • Sequence Homology, Nucleic Acid

Substances

  • Bacterial Proteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases