RT Journal Article
SR Electronic
T1 Recombineering in <em>C. elegans</em>: genome editing using <em>in vivo</em> assembly of linear DNAs
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 050815
DO 10.1101/050815
A1 Alexandre Paix
A1 Helen Schmidt
A1 Geraldine Seydoux
YR 2016
UL http://biorxiv.org/content/early/2016/04/28/050815.abstract
AB Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate that homology-dependent repair (HDR) is robust in C. elegans using linear templates with short homologies (~35 bases). Templates with homology to only one side of a double-strand break initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments precisely to DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.