RRC ID 30616
著者 Ansai S, Kinoshita M.
タイトル Targeted mutagenesis using CRISPR/Cas system in medaka.
ジャーナル Biol Open
Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5' end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka.
巻・号 3(5)
ページ 362-71
公開日 2014-4-11
DOI 10.1242/bio.20148177
PII bio.20148177
PMID 24728957
PMC PMC4021358
IF 2.029
引用数 82
WOS 分野 BIOLOGY
リソース情報
メダカ database d-rR/TOKYO (MT837)