First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts
The current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome dat...
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MDPI AG
2021-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/1/396 |
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| author | Sapna Badhan Andrew S. Ball Nitin Mantri |
| author_facet | Sapna Badhan Andrew S. Ball Nitin Mantri |
| author_sort | Sapna Badhan |
| collection | DOAJ |
| description | The current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome data, CRISPR/Cas9 presents a massive opportunity for plant breeders and researchers. The successful delivery of ribonucleoproteins (RNPs), which are composed of Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) and are used in combination with various transformation methods or lately available novel nanoparticle-based delivery approaches, allows targeted mutagenesis in plants species. Even though this editing technique is limitless, it has still not been employed in many plant species to date. Chickpea is the second most crucial winter grain crop cultivated worldwide; there are currently no reports on CRISPR/Cas9 gene editing in chickpea. Here, we selected the <i>4-coumarate ligase</i> (<i>4CL</i>) and <i>Reveille 7</i> (<i>RVE7</i>) genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The <i>4CL</i> represents a key enzyme involved in phenylpropanoid metabolism in the lignin biosynthesis pathway. It regulates the accumulation of lignin under stress conditions in several plants. The <i>RVE7</i> is a MYB transcription factor which is part of regulating circadian rhythm in plants. The knockout of these selected genes in the chickpea protoplast using DNA-free CRISPR/Cas9 editing represents a novel approach for achieving targeted mutagenesis in chickpea. Results showed high-efficiency editing was achieved for <i>RVE7</i> gene in vivo compared to the <i>4CL</i> gene. This study will help unravel the role of these genes under drought stress and understand the complex drought stress mechanism pathways. This is the first study in chickpea protoplast utilizing CRISPR/Cas9 DNA free gene editing of drought tolerance associated genes. |
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| last_indexed | 2024-03-10T13:34:17Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-d3cdcaf7df4f4e70abeefbebca3a7a302023-11-21T07:42:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-01-0122139610.3390/ijms22010396First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea ProtoplastsSapna Badhan0Andrew S. Ball1Nitin Mantri2The Pangenomics Lab, School of Science, RMIT University, Melbourne 3000, AustraliaThe Pangenomics Lab, School of Science, RMIT University, Melbourne 3000, AustraliaThe Pangenomics Lab, School of Science, RMIT University, Melbourne 3000, AustraliaThe current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome data, CRISPR/Cas9 presents a massive opportunity for plant breeders and researchers. The successful delivery of ribonucleoproteins (RNPs), which are composed of Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) and are used in combination with various transformation methods or lately available novel nanoparticle-based delivery approaches, allows targeted mutagenesis in plants species. Even though this editing technique is limitless, it has still not been employed in many plant species to date. Chickpea is the second most crucial winter grain crop cultivated worldwide; there are currently no reports on CRISPR/Cas9 gene editing in chickpea. Here, we selected the <i>4-coumarate ligase</i> (<i>4CL</i>) and <i>Reveille 7</i> (<i>RVE7</i>) genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The <i>4CL</i> represents a key enzyme involved in phenylpropanoid metabolism in the lignin biosynthesis pathway. It regulates the accumulation of lignin under stress conditions in several plants. The <i>RVE7</i> is a MYB transcription factor which is part of regulating circadian rhythm in plants. The knockout of these selected genes in the chickpea protoplast using DNA-free CRISPR/Cas9 editing represents a novel approach for achieving targeted mutagenesis in chickpea. Results showed high-efficiency editing was achieved for <i>RVE7</i> gene in vivo compared to the <i>4CL</i> gene. This study will help unravel the role of these genes under drought stress and understand the complex drought stress mechanism pathways. This is the first study in chickpea protoplast utilizing CRISPR/Cas9 DNA free gene editing of drought tolerance associated genes.https://www.mdpi.com/1422-0067/22/1/396CRISPR/Cas9chickpeaDNA free gene editingprotoplast<i>4-coumarate ligase</i> (<i>4CL</i>)<i>Reveille 7</i> (<i>RVE7</i>) |
| spellingShingle | Sapna Badhan Andrew S. Ball Nitin Mantri First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts International Journal of Molecular Sciences CRISPR/Cas9 chickpea DNA free gene editing protoplast <i>4-coumarate ligase</i> (<i>4CL</i>) <i>Reveille 7</i> (<i>RVE7</i>) |
| title | First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts |
| title_full | First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts |
| title_fullStr | First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts |
| title_full_unstemmed | First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts |
| title_short | First Report of CRISPR/Cas9 Mediated DNA-Free Editing of <i>4CL</i> and <i>RVE7</i> Genes in Chickpea Protoplasts |
| title_sort | first report of crispr cas9 mediated dna free editing of i 4cl i and i rve7 i genes in chickpea protoplasts |
| topic | CRISPR/Cas9 chickpea DNA free gene editing protoplast <i>4-coumarate ligase</i> (<i>4CL</i>) <i>Reveille 7</i> (<i>RVE7</i>) |
| url | https://www.mdpi.com/1422-0067/22/1/396 |
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