By Bio-tech International Institute
Introduction
CRISPR/Cas9 technology is a powerful tool for genome editing that allows precise modifications to specific genes within an organism's DNA. In cannabis (Cannabis sativa), CRISPR/Cas9-mediated editing has potential applications for improving traits such as cannabinoid production, pest resistance, and growth characteristics. The PHYTOENE DESATURASE (PDS) gene is commonly used as a reporter gene in plant genetic studies because its disruption results in a visible phenotype—bleaching of plant tissues due to impaired carotenoid biosynthesis.
Understanding CRISPR/Cas9
In the ever-evolving field of plant biotechnology, the CRISPR/Cas9 system has emerged as a groundbreaking tool for precise genome editing. This technology is now being applied to cannabis plants, targeting the PHYTOENE DESATURASE (PDS) gene, which plays a crucial role in carotenoid biosynthesis. The implications of this research are vast, promising advancements in scientific understanding and agricultural practices.
CRISPR/Cas9, short for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9, is a revolutionary genome-editing technology. It allows scientists to make precise, targeted changes to the DNA of living organisms. The system consists of two.
Main components:
Cas9 Protein: An enzyme that acts like molecular scissors, cutting DNA at a specific location.
Guide RNA (gRNA): A custom RNA sequence that directs Cas9 to the target DNA sequence.
Targeting the PDS Gene in Cannabis
The PDS gene is integral to the production of carotenoids, essential for chlorophyll synthesis and photosynthesis. Disrupting this gene results in an albino phenotype characterized by white or pale leaves due to the absence of chlorophyll. This visible change makes the PDS gene an excellent marker for confirming successful gene editing.
Objective
This study aims to explore the feasibility and efficiency of CRISPR/Cas9-mediated editing of the PDS gene in Cannabis sativa. This research seeks to establish a proof-of-concept for gene editing in cannabis, which could pave the way for further genetic modifications to enhance desirable traits such as cannabinoid production, disease resistance, and growth characteristics.
Materials and Methods
Plant Material: Cannabis sativa seedlings were grown under controlled environmental conditions, and young, healthy plants were selected for transformation.
CRISPR/Cas9 Construct Design:
- A CRISPR/Cas9 system targeting the PDS gene was constructed using a single-guide RNA (sgRNA) designed to target a conserved region of the PDS gene.
- The sgRNA was cloned into a CRISPR/Cas9 expression vector, which also contained a Cas9 nuclease and a selectable marker for plant transformation.
Transformation and Regeneration:
- Cannabis explants (leaf or stem sections) were transformed with the CRISPR/Cas9 construct using Agrobacterium-mediated transformation.
- Transformed explants were cultured on selective media containing antibiotics to promote the growth of transformed cells and suppress untransformed cells.
- Regeneration protocols were optimized to encourage the development of shoots and roots from the transformed explants.
Screening and Confirmation:
- Successfully regenerated plants were screened for the presence of the
CRISPR/Cas9 construct using PCR.
- Edited plants were analyzed for mutations in the PDS gene using sequencing.
- Phenotypic screening for leaf bleaching was conducted as an initial indicator of successful PDS gene disruption.
Results
Transformation Efficiency: Transformation efficiency varied, with the successful integration of the CRISPR/Cas9 construct observed in a subset of regenerated plants.
Mutation Analysis: Sequencing of the PDS gene in edited plants confirmed the presence of insertions and deletions (indels) at the targeted site, demonstrating successful editing. The albino phenotype was observed in several edited plants, confirming the successful knockout of the PDS gene.
Phenotypic Changes: Edited plants exhibited the expected bleaching phenotype in leaves, indicative of disrupted carotenoid biosynthesis due to PDS gene knockout.
Challenges and Future Directions
Transformation Efficiency: While CRISPR/Cas9-mediated editing was successful, transformation efficiency remains a limiting factor. Optimization of transformation and regeneration protocols specific to cannabis is needed to improve outcomes.
Off-Target Effects: Future studies should assess the potential off-target effects of CRISPR/Cas9 editing in cannabis to ensure precision and safety in genetic modifications.
Applications: The methods developed here can be applied to edit other genes in cannabis to enhance desirable traits, paving the way for precision breeding and developing improved cannabis varieties.
Discussion
The successful editing of the PDS gene in cannabis sativa demonstrates the potential of CRISPR/Cas9 technology for genetic modification in this species. The observed albino phenotype serves as a clear marker for gene editing, facilitating the identification of successful edits. This study lays the groundwork for future research aimed at enhancing specific traits in cannabis through targeted gene editing.
Conclusion
CRISPR/Cas9-mediated editing of the PDS gene in cannabis demonstrates the potential of this technology for precise genetic modifications in cannabis. The approach provides a platform for future genetic studies and the development of novel cannabis strains with enhanced traits.
Future Directions
Future research could focus on:
- Targeting other genes related to cannabinoid biosynthesis to enhance the production of specific cannabinoids.
- Improving transformation and regeneration protocols to increase efficiency.
- Investigating the off-target effects of CRISPR/Cas9 editing in cannabis.
By leveraging CRISPR/Cas9 technology, researchers can unlock new possibilities in cannabis genetics, leading to improved cultivars with enhanced traits.
Source: 7/29/2024
: Pavese et al., 2021. CRISPR/Cas9 mediated editing of phytoene desaturase gene in plants Springer.
(1) CRISPR/Cas9 mediated editing of phytoene desaturase gene in ... - Springer. https://link.springer.com/content/pdf/10.1007/s13562-023-00866-w.pdf.
(2) Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene .... https://www.nature.com/articles/s41598-022-21566-w.pdf.
(3) CRISPR/Cas9 mediated editing of phytoene desaturase gene in squash .... https://link.springer.com/article/10.1007/s13562-023-00866-w.
(4) Frontiers | CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in .... https://www.frontiersin.org/journals/plant-
science/articles/10.3389/fpls.2023.1226911/full.
(5) Precision genome editing in plants: state-of-the-art in CRISPR/Cas9 .... https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02385-5.
(6) Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants. https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.902413/full.
(7) CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for .... https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.589517/full.
(8) Editorial: Methods and applications of CRISPR technology in plant .... https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1207257/full.
(9) undefined. https://doi.org/10.3389/fpls.2022.902413.
