期刊
GENETICS
卷 201, 期 1, 页码 47-+出版社
GENETICS SOCIETY AMERICA
DOI: 10.1534/genetics.115.179382
关键词
CRISPR-Cas9; genome editing; homology-directed repair; ribonucleoprotein complexes; C. elegans
资金
- National Institutes of Health Office of Research Infrastructure Programs [P40 OD010440]
- National Institutes of Health [R01HD37047]
Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.
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