Targeted mutagenesis using CRISPR/Cas system in medaka
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, Oryz...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
The Company of Biologists
2014-04-01
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| Series: | Biology Open |
| Subjects: | |
| Online Access: | http://bio.biologists.org/content/3/5/362 |
| _version_ | 1818859734923476992 |
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| author | Satoshi Ansai Masato Kinoshita |
| author_facet | Satoshi Ansai Masato Kinoshita |
| author_sort | Satoshi Ansai |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-19T09:16:54Z |
| format | Article |
| id | doaj.art-981af22ddbf0444e9356e8de6a967002 |
| institution | Directory Open Access Journal |
| issn | 2046-6390 |
| language | English |
| last_indexed | 2024-12-19T09:16:54Z |
| publishDate | 2014-04-01 |
| publisher | The Company of Biologists |
| record_format | Article |
| series | Biology Open |
| spelling | doaj.art-981af22ddbf0444e9356e8de6a9670022022-12-21T20:28:05ZengThe Company of BiologistsBiology Open2046-63902014-04-013536237110.1242/bio.2014817720148177Targeted mutagenesis using CRISPR/Cas system in medakaSatoshi AnsaiMasato KinoshitaClustered 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.http://bio.biologists.org/content/3/5/362MedakaCRISPR/CasGenome editingMutagenesisOff-target alterations |
| spellingShingle | Satoshi Ansai Masato Kinoshita Targeted mutagenesis using CRISPR/Cas system in medaka Biology Open Medaka CRISPR/Cas Genome editing Mutagenesis Off-target alterations |
| title | Targeted mutagenesis using CRISPR/Cas system in medaka |
| title_full | Targeted mutagenesis using CRISPR/Cas system in medaka |
| title_fullStr | Targeted mutagenesis using CRISPR/Cas system in medaka |
| title_full_unstemmed | Targeted mutagenesis using CRISPR/Cas system in medaka |
| title_short | Targeted mutagenesis using CRISPR/Cas system in medaka |
| title_sort | targeted mutagenesis using crispr cas system in medaka |
| topic | Medaka CRISPR/Cas Genome editing Mutagenesis Off-target alterations |
| url | http://bio.biologists.org/content/3/5/362 |
| work_keys_str_mv | AT satoshiansai targetedmutagenesisusingcrisprcassysteminmedaka AT masatokinoshita targetedmutagenesisusingcrisprcassysteminmedaka |