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

  1. Geraldine Seydouxa,1
  1. aDepartment of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore MD 21205
  1. Contributed by Geraldine Seydoux, October 26, 2017 (sent for review July 5, 2017; reviewed by Dana Carroll and James E. Haber)

Significance

Genome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing in mammalian cells that takes advantage of an efficient mechanism for gene conversion that utilizes linear donors. We demonstrate that PCR fragments containing edits up to 1 kb require only 35-bp homology sequences to initiate repair of Cas9-induced double-stranded breaks in human cells and mouse embryos. We experimentally determine donor DNA design rules that maximize the recovery of edits without cloning or selection.

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.

Footnotes

  • ?1To whom correspondence may be addressed. Email: apaix1{at}jhmi.edu or gseydoux{at}jhmi.edu.
  • Author contributions: A.P. and G.S. designed research; A.P., A.F., D.H.G., H.K., M.J.G., D.R., and S.P. performed research; A.P., A.F., D.H.G., and G.S. analyzed data; and A.P., A.F., D.H.G., R.G., R.R.R., and G.S. wrote the paper.

  • Reviewers: D.C., University of Utah; and J.E.H., Brandeis University.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1711979114/-/DCSupplemental.

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