As in other organisms, CRISPR-Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost-effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks. Here, I describe a methodology in C. elegans to efficiently knock-in epitope tags in 8-9 days, using a temperature sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 basepairs of homology drive efficient knock-in of point mutations, and that disabling non-homologous end-joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35-80 basepairs are sufficient for efficient editing and double-strand breaks up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.
- non-homologous end-joining
- oligonucleotide-mediated homologous recombination
- Received November 3, 2014.
- Accepted December 8, 2014.
- Copyright © 2014, The Genetics Society of America