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

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ID: 307624
2015
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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.
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openalex_W2120998017 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Alexandre Paix, Andrew W. Folkmann, Dominique Rasoloson, Géraldine Seydoux
Journal current genetics
Year 2015
DOI
10.1534/genetics.115.179382
URL
https://www.genetics.org/content/genetics/201/1/47.full.pdf
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