Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans

Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23.


Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30-60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.

Keywords: CRISPR; Caenorhabditis elegans; Cas9; genome editing; homology-directed repair; short homology arms.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Associated Proteins / metabolism*
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / metabolism*
  • Codon, Terminator
  • DNA Breaks, Double-Stranded
  • Gene Deletion
  • Gene Expression
  • Gene Targeting / methods*
  • Genes, Reporter
  • Genome*
  • Homologous Recombination
  • Mutagenesis, Insertional
  • Oligonucleotides
  • Recombinational DNA Repair


  • CRISPR-Associated Proteins
  • Codon, Terminator
  • Oligonucleotides