New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i
Abstract Background For ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | Plant Methods |
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| Online Access: | https://doi.org/10.1186/s13007-024-01173-8 |
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| author | Jérémy Villette Fatma Lecourieux Eliot Bastiancig Marie-Claire Héloir Benoit Poinssot |
| author_facet | Jérémy Villette Fatma Lecourieux Eliot Bastiancig Marie-Claire Héloir Benoit Poinssot |
| author_sort | Jérémy Villette |
| collection | DOAJ |
| description | Abstract Background For ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. ‘Chardonnay’). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy. Results By comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. ‘Chardonnay’. These results are directly correlated with Cas9 expression level. Conclusions Taken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine. |
| first_indexed | 2024-04-24T19:56:12Z |
| format | Article |
| id | doaj.art-a881c9ee298c4759940e5475b4c51cc5 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-04-24T19:56:12Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-a881c9ee298c4759940e5475b4c51cc52024-03-24T12:21:47ZengBMCPlant Methods1746-48112024-03-0120111410.1186/s13007-024-01173-8New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9iJérémy Villette0Fatma Lecourieux1Eliot Bastiancig2Marie-Claire Héloir3Benoit Poinssot4Agroécologie, INRAE, Institut Agro, Université de BourgogneUMR1287 EGFV, CNRS, Université de Bordeaux, INRAE, Bordeaux Sciences Agro, ISVVAgroécologie, INRAE, Institut Agro, Université de BourgogneAgroécologie, INRAE, Institut Agro, Université de BourgogneAgroécologie, INRAE, Institut Agro, Université de BourgogneAbstract Background For ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. ‘Chardonnay’). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy. Results By comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. ‘Chardonnay’. These results are directly correlated with Cas9 expression level. Conclusions Taken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.https://doi.org/10.1186/s13007-024-01173-8Vitis viniferaCRISPR/cas9Efficient gene editingzCas9i |
| spellingShingle | Jérémy Villette Fatma Lecourieux Eliot Bastiancig Marie-Claire Héloir Benoit Poinssot New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i Plant Methods Vitis vinifera CRISPR/cas9 Efficient gene editing zCas9i |
| title | New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i |
| title_full | New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i |
| title_fullStr | New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i |
| title_full_unstemmed | New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i |
| title_short | New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i |
| title_sort | new improvements in grapevine genome editing high efficiency biallelic homozygous knock out from regenerated plantlets by using an optimized zcas9i |
| topic | Vitis vinifera CRISPR/cas9 Efficient gene editing zCas9i |
| url | https://doi.org/10.1186/s13007-024-01173-8 |
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