High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9ribonucleoprotein 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 vifro-assembled Cas9-CRISPR RNA (crRNA) frans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabdifis 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, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.