High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Issue 1 (17th July 2015)

• Record Type: Journal Article
• Authors: Paix, Alexandre; Folkmann, Andrew; Rasoloson, Dominique; Seydoux, Geraldine
• Is Part Of: Genetics. Volume 201:Issue 1(2015)
• Page Start: 47
• Page End: 54
• Publication Date: 2015-07-17
• Subjects: CRISPR-Cas9 -- genome editing -- homology-directed repair -- ribonucleoprotein complexes -- C. elegans; Genetics -- Periodicals; 576.5
• DOI: 10.1534/genetics.115.179382
• Languages: English
• ISSNs: 0016-6731

Abstract

Abstract: 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, 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.