Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the Caenorhabditis elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4-5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications, and will provide researchers with increased flexibility when designing genome editing experiments.
- Received July 9, 2015.
- Accepted July 30, 2015.
- Copyright © 2015, The Genetics Society of America