TY - JOUR
T1 - Cas9-assisted recombineering in C. elegans
T2 - Genome editing using in vivo assembly of linear DNAs
AU - Paix, Alexandre
AU - Schmidt, Helen
AU - Seydoux, Geraldine
N1 - Funding Information:
National Institutes of Health (NIH) [R01HD37047]. G.S. is an investigator of the Howard Hughes Medical Institute. Some strains were provided by or deposited at the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs [P40 OD010440]. Funding for open access charge: HHMI.
Publisher Copyright:
© 2016 The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2016/9/6
Y1 - 2016/9/6
N2 - 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, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) 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 for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise 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.
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, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) 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 for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise 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.
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U2 - 10.1093/nar/gkw502
DO - 10.1093/nar/gkw502
M3 - Article
C2 - 27257074
AN - SCOPUS:84988423394
SN - 0305-1048
VL - 44
SP - e128
JO - Nucleic acids research
JF - Nucleic acids research
IS - 15
ER -