Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes

Genetics. 2015 Oct;201(2):449-58. doi: 10.1534/genetics.115.180679. Epub 2015 Jul 30.


Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. 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.

Keywords: C. elegans; CRISPR; Cas9; genome editing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Caenorhabditis elegans / genetics*
  • DNA Breaks, Double-Stranded
  • Gene Targeting
  • Genome*
  • Genotype
  • Homologous Recombination / genetics
  • RNA Editing / genetics*
  • Recombinational DNA Repair / genetics