Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

被引：203

作者：

Paix, Alexandre ^{[1
]}

Wang, Yuemeng ^{[1
]}

Smith, Harold E. ^{[2
]}

Lee, Chih-Yung S. ^{[1
]}

Calidas, Deepika ^{[1
]}

Lu, Tu ^{[1
]}

Smith, Jarrett ^{[1
]}

Schmidt, Helen ^{[1
]}

Krause, Michael W. ^{[2
]}

Seydoux, Geraldine ^{[1
]}

机构：

[1] Johns Hopkins Univ, Sch Med, Dept Mol Biol & Genet, Baltimore, MD 21205 USA

[2] NIDDK, NIH, Bethesda, MD 20892 USA

来源：

GENETICS | 2014年 / 198卷 / 04期

基金：

美国国家卫生研究院;

关键词：

C-ELEGANS; CRISPR/CAS9;

D O I：

10.1534/genetics.114.170423

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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 (similar to 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.

引用

页码：1347 / +

页数：25

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