Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans
| Autor: | Chih Yung S. Lee, Alexandre Paix, Harold E. Smith, Jarrett Smith, Deepika Calidas, Yuemeng Wang, Michael W. Krause, Helen Schmidt, Tu Lu, Geraldine Seydoux |
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| Rok vydání: | 2014 |
| Předmět: |
CRISPR-Associated Proteins
Oligonucleotides Gene Expression Investigations Biology Genome Homology directed repair 03 medical and health sciences 0302 clinical medicine Genome editing Genes Reporter Genetics genome editing Animals CRISPR DNA Breaks Double-Stranded Cas9 Caenorhabditis elegans Homologous Recombination Gene 030304 developmental biology 0303 health sciences Methods Technology and Resources Recombinational DNA Repair biology.organism_classification Non-homologous end joining homology-directed repair Mutagenesis Insertional Gene Targeting Codon Terminator short homology arms Gene Deletion 030217 neurology & neurosurgery |
| Zdroj: | Genetics |
| ISSN: | 1943-2631 |
| DOI: | 10.1534/genetics.114.170423 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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