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

Alexandre Paix, Andrew Folkmann, Daniel H. Goldman, Heather Kulaga, Michael J. Grzelak, Dominique Rasoloson, Supriya Paidemarry, Rachel Green, Randall R. Reed, Geraldine Seydoux

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)E10745-E10754
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number50
DOIs
StatePublished - Dec 12 2017

Fingerprint

DNA Breaks
DNA
Nucleotides
Genome
Gene Conversion
Single-Stranded DNA
Deoxyribonuclease I
Embryonic Structures
RNA
Polymerase Chain Reaction
Mutation
Gene Editing
Proteins

Keywords

  • CRISPR
  • HDR
  • PCR repair template
  • SDSA
  • Short homology arms

ASJC Scopus subject areas

  • General

Cite this

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. / Paix, Alexandre; Folkmann, Andrew; Goldman, Daniel H.; Kulaga, Heather; Grzelak, Michael J.; Rasoloson, Dominique; Paidemarry, Supriya; Green, Rachel; Reed, Randall R.; Seydoux, Geraldine.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 50, 12.12.2017, p. E10745-E10754.

Research output: Contribution to journalArticle

Paix, Alexandre ; Folkmann, Andrew ; Goldman, Daniel H. ; Kulaga, Heather ; Grzelak, Michael J. ; Rasoloson, Dominique ; Paidemarry, Supriya ; Green, Rachel ; Reed, Randall R. ; Seydoux, Geraldine. / Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. In: Proceedings of the National Academy of Sciences of the United States of America. 2017 ; Vol. 114, No. 50. pp. E10745-E10754.
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