High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9ribonucleoprotein complexes

Alexandre Paix, Andrew Folkmann, Dominique Rasoloson, Geraldine Seydoux

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)47-54
Number of pages8
JournalGenetics
Volume201
Issue number1
DOIs
StatePublished - Sep 1 2015

Keywords

  • C. elegans
  • CRISPR-Cas9
  • Genome editing
  • Homology-directed repair
  • Ribonucleoprotein complexes

ASJC Scopus subject areas

  • Genetics

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