Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans. Issue 4 (23rd September 2014)

• Record Type: Journal Article
• Authors: Paix, Alexandre; Wang, Yuemeng; Smith, Harold E; Lee, Chih-Yung S; Calidas, Deepika; Lu, Tu; Smith, Jarrett; Schmidt, Helen; Krause, Michael W; Seydoux, Geraldine
• Is Part Of: Genetics. Volume 198:Issue 4(2014)
• Page Start: 1347
• Page End: 1356
• Publication Date: 2014-09-23
• Subjects: CRISPR -- Cas9 -- genome editing -- homology-directed repair -- short homology arms -- Caenorhabditis elegans; Genetics -- Periodicals; 576.5
• DOI: 10.1534/genetics.114.170423
• Languages: English
• ISSNs: 0016-6731

Abstract

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.