Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells
Abstract Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-through...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-02-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-024-55088-4 |
| _version_ | 1797275044306485248 |
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| author | Ellen Slaman Lisanne Kottenhagen William de Martines Gerco C. Angenent Ruud A. de Maagd |
| author_facet | Ellen Slaman Lisanne Kottenhagen William de Martines Gerco C. Angenent Ruud A. de Maagd |
| author_sort | Ellen Slaman |
| collection | DOAJ |
| description | Abstract Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing. |
| first_indexed | 2024-03-07T15:07:45Z |
| format | Article |
| id | doaj.art-f946cc8f449147b18c48df0842134f75 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-07T15:07:45Z |
| publishDate | 2024-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-f946cc8f449147b18c48df0842134f752024-03-05T18:50:33ZengNature PortfolioScientific Reports2045-23222024-02-0114111310.1038/s41598-024-55088-4Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cellsEllen Slaman0Lisanne Kottenhagen1William de Martines2Gerco C. Angenent3Ruud A. de Maagd4Laboratory of Molecular Biology, Wageningen University & ResearchLaboratory of Molecular Biology, Wageningen University & ResearchLaboratory of Molecular Biology, Wageningen University & ResearchLaboratory of Molecular Biology, Wageningen University & ResearchBioscience, Wageningen University & ResearchAbstract Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.https://doi.org/10.1038/s41598-024-55088-4 |
| spellingShingle | Ellen Slaman Lisanne Kottenhagen William de Martines Gerco C. Angenent Ruud A. de Maagd Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells Scientific Reports |
| title | Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells |
| title_full | Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells |
| title_fullStr | Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells |
| title_full_unstemmed | Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells |
| title_short | Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells |
| title_sort | comparison of cas12a and cas9 mediated mutagenesis in tomato cells |
| url | https://doi.org/10.1038/s41598-024-55088-4 |
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