Abstract
ABSTRACTClustered 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.
Highlights
Genome editing with artificial nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has become a powerful tool for approaches involving reverse genetics in a wide range of organisms (Carroll, 2011; Joung and Sander, 2013)
To induce efficient expression of Cas9 nuclease, we generated a pCS2+ -based Cas9 nuclease expression vector to produce a capped RNA by SP6 RNA polymerase, containing a human codon-optimized S. pyogenes Cas9 nuclease fused to a triple FLAG tag and two nuclear localization signal (NLS) in both N- and C-terminals previously used in cultured human cells (Cong et al, 2013; Ran et al, 2013b)
All the sequenced clones had altered sequences, including 6 types of mutations in embryo #1-1 (17 of 17 sequenced clones; 100%) (Fig. 1D) and 7 types of mutations in embryo #1-2 (16 of 16; 100%) (Fig. 1E). These results indicate that the RNA-guided endonuclease (RGEN) introduced DNA double-strand breaks at the target genomic sequence and thereby induced indels via error-prone nonhomologous end joining repair with high efficiency
Summary
Genome editing with artificial nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has become a powerful tool for approaches involving reverse genetics in a wide range of organisms (Carroll, 2011; Joung and Sander, 2013). These enzymes efficiently induce site-specific DNA double-strand breaks (DSBs), resulting in targeted gene disruptions by insertions and deletions (indels) or targeted gene integrations by homologous recombination. CRISPR and Cas proteins are essential components of the adaptive immune system in bacteria and Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
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