High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes

Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method,…

• HH
  Howard Hughes Medical Institute
• NI
  National Institutes of Health
• OO
  Office of Research Infrastructure Programs, National Institutes of Health