Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 sites in Caenorhabditis elegans

Alexandre Paix, Yuemeng Wang, Harold E. Smith, Chih-Yung Lee, Deepika Calidas, Tu Lu, Jarrett Smith, Helen Schmidt, Michael W. Krause, Geraldine Seydoux

Research output: Contribution to journalArticle

Abstract

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (~30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.

Original languageEnglish (US)
Pages (from-to)1347-1356
Number of pages10
JournalGenetics
Volume198
Issue number4
DOIs
StatePublished - Dec 1 2014

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CRISPR-Associated Proteins
Caenorhabditis elegans
DNA
Genome
Genes
Double-Stranded DNA Breaks
Gene Deletion
Peptides
Mutation
Gene Editing
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 sites in Caenorhabditis elegans. / Paix, Alexandre; Wang, Yuemeng; Smith, Harold E.; Lee, Chih-Yung; Calidas, Deepika; Lu, Tu; Smith, Jarrett; Schmidt, Helen; Krause, Michael W.; Seydoux, Geraldine.

In: Genetics, Vol. 198, No. 4, 01.12.2014, p. 1347-1356.

Research output: Contribution to journalArticle

Paix, Alexandre ; Wang, Yuemeng ; Smith, Harold E. ; Lee, Chih-Yung ; Calidas, Deepika ; Lu, Tu ; Smith, Jarrett ; Schmidt, Helen ; Krause, Michael W. ; Seydoux, Geraldine. / Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 sites in Caenorhabditis elegans. In: Genetics. 2014 ; Vol. 198, No. 4. pp. 1347-1356.
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