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 S. Lee, Deepika Calidas, Tu Lu, Jarrett Smith, Helen Schmidt, Michael W. Krause, Geraldine Seydoux

Research output: Contribution to journalArticlepeer-review

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

ASJC Scopus subject areas

  • Genetics

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