First report of CRISPR/cas9 mediated dna-free editing of 4cl and rve7 genes in chickpea protoplasts
Open accessAbstract
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 4-coumarate ligase (4CL) and Reveille 7 (RVE7) genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The 4CL 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 RVE7 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 RVE7 gene in vivo compared to the 4CL 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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Successful editing of the 4CL and RVE7 genes in chickpea protoplasts demonstrates potential for improving drought tolerance.
“In leguminous crops, successful editing of the 4CL and RVE7 genes in chickpea protoplasts has demonstrated potential for improving drought tolerance (Badhan et al., 2021).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Badhan et al., 2021
The Results (§2.4; page unavailable) report substantial editing of RVE7, but only one 4CL sample showed a 2% knockout score; the Discussion (§3) says that result may be a sequencing artefact. The Conclusions (§4) describe drought-tolerance improvement as a potential future application, not an outcome demonstrated in plants. (LLM, conf=0.87)
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- Authors
- Badhan, S., Ball, A. S., & Mantri, N.
- Affiliations
- RMIT University (AU)
- Corresponding author
- Mantri, Nitin
- Venue
- International Journal of Molecular Sciences
- Publisher
- MDPI AG
- Published
- 2021-01-01
- Volume
- 22
- Issue
- 1
- Pages
- 396
- ISSN
- 1422-0067 (ISSN-L)
- PMC
- PMC7795094
- OpenAlex
- W3115890091
- Cited by
- 193
- References
- 71
- Citations by year
- 2026: 23 · 2025: 51 · 2024: 33 · 2023: 42 · 2022: 36 · 2021: 8
- FWCI
- 9.34
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- International Journal of Molecular Sciences (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RMIT Research Repository (RMIT University Library) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetic and Environmental Crop Studies
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Keywords
- CRISPR/Cas9, chickpea, protoplast transformation, MYB transcription factors, drought tolerance, phenylpropanoid metabolism, lignin biosynthesis, circadian rhythm, targeted mutagenesis, single guide RNA
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Coenzyme A Ligases, Lignin, Plant Proteins, Stress, Physiological, Transcription Factors, Cicer, Plants, Genetically Modified, Gene Knockout Techniques, Droughts
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