RT Journal Article SR Electronic T1 Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks JF bioRxiv FD Cold Spring Harbor Laboratory SP 161109 DO 10.1101/161109 A1 Paix, Alexandre A1 Folkmann, Andrew A1 Goldman, Daniel H A1 Kulaga, Heather A1 Grzelak, Michael A1 Rasoloson, Dominique A1 Paidemarry, Supriya A1 Green, Rachel A1 Reed, Randall A1 Seydoux, Geraldine YR 2017 UL http://biorxiv.org/content/early/2017/07/09/161109.abstract AB The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in 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 1000 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 (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.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 that takes advantage of an efficient mechanism for DNA repair called synthesis-dependent strand annealing. We demonstrate that synthetic linear DNAs (ssODNs and PCR fragments) with ∼35bp homology arms function as efficient donors for SDSA repair of Cas9-induced double-strand breaks. Edits from 1 to 1000 base pairs can be introduced in the genome without cloning or selection.