In:
Genetics, Oxford University Press (OUP), Vol. 198, No. 4 ( 2014-12-01), p. 1347-1356
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.
Type of Medium:
Online Resource
ISSN:
1943-2631
DOI:
10.1534/genetics.114.170423
Language:
English
Publisher:
Oxford University Press (OUP)
Publication Date:
2014
detail.hit.zdb_id:
1477228-0
SSG:
12
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