by Ashok Karuppannasamy, Yogi Dhawane and Asokan Ramasamy
The field of insect genome editing is transforming, and the shift from Cas9 to Cas12a is at the heart of it. The demonstration of CRISPR/Cas9-mediated editing in Bactrocera dorsalis represents a leap forward in understanding insect genetics. Ashok et al. (2023) edited an eye colour gene in B. dorsalis which resulted in a white eye colour phenotype (Figure 1). These visible alterations confirmed the efficacy of the CRISPR/Cas9 RNP-mediated editing and underscored its potential for functional genomic studies in non-model insects. However, in our recent study (Yogi et al., 2024), we unveiled a ground breaking advancement by harnessing the potential of CRISPR/Cas12a in a globally important agricultural pest, Spodoptera frugiperda - a non-model organism. This work represents a paradigm shift in insect molecular biology, promising new horizons for functional genomics and sustainable pest management.
Why Cas12a?
CRISPR/Cas9 has been the go-to genome editing tool for years. However, Cas12a brings several unique advantages. Unlike Cas9, which requires a GC-rich protospacer adjacent motif (PAM), Cas12a can target T-rich PAMs. This feature greatly expands the range of target sites, especially in genomes with limited GC content. Cas12a causes staggered cuts in DNA, which makes it ideal for precision edits and multiplexing-editing multiple genes simultaneously.
The First Cas12a Application in non-model organism Spodoptera frugiperda
Our study is the first to successfully apply Cas12a in Spodoptera frugiperda, an invasive pest that defies most current management strategies. We targeted the tryptophan 2,3-dioxygenase gene, a crucial gene linked to eye colour. By microinjecting Cas12a ribonucleoprotein complexes (RNPs) into pre-blastoderm (G0) eggs, we achieved precise edits with deletions and insertions. The mutants exhibited phenotypic Validations with varied eye-colour phenotypes, which we further validated through in silico protein modeling to understand the underlying changes at the molecular level (Figure 2). This proof-of-concept underscores Cas12a’s utility for functional genomics in non-model insects.
Paving the way for genetic biocontrol
One of the most promising applications of genome editing in pest management is the precision-guided Sterile Insect Technique (pgSIT). Previously demonstrated in Drosophila melanogaster, this method holds tremendous potential for pests like S. frugiperda. By validating Cas12a in this species, we’ve laid the groundwork for developing advanced genetic biocontrol tools, including:
Multiplexed Gene Targeting: Editing multiple genes simultaneously to disrupt pest fitness.
Refined Biocontrol Strategies: Adapting pgSIT to the reproductive and ecological traits of agricultural pests.
Controllable Gene Drives: Designing gene drives to ensure population suppression in a controlled and reversible manner.
Looking Ahead
This research not only marks a breakthrough in applying Cas12a to non-model insects but also sets the stage for broader applications in pest management. Moving forward, we aim to:
Ø Explore additional target genes to assess Cas12a's versatility.
Ø Develop multiplexing approaches for complex gene networks.
Ø Tailor biocontrol methods to align with ecological sustainability.
Conclusion
The leap from Cas9 to Cas12a is more than a technical upgrade. It is a revolution in how we approach insect genome editing. By unlocking new possibilities for precise, efficient, and sustainable pest management, we are taking a significant step toward addressing global agricultural challenges. The future is bright, and it’s time to embrace the power of “12” for a transformative impact in entomology.
For more details, please refer:
Ashok, K., Bhargava, C.N., Asokan, R.*, Pradeep, C., Kennedy, J.S., Rai, A. and Manamohan, M., 2023. First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel)(Tephritidae: Diptera). Archives of Insect Biochemistry and Physiology, 113(4), p.e22024.
Yogi, D., Ashok, K.*, Anu, C.N., Shashikala, T., Pradeep, C., Bhargava, C.N., Parvathy, M.S., Jithesh, M.N., Manamohan, M., Jha, G.K. and Asokan, R.* (2024). CRISPR/Cas12a ribonucleoprotein mediated editing of tryptophan 2, 3-dioxygenase of Spodoptera frugiperda. Transgenic Research, pp.1-13.
Ashok Karuppannasamy is one of the Associate Editors of Indian Entomologist. He works as a Post Doctoral Fellow, at Tata Institute for Genetics and Society, InStem Building, NCBS Campus, Bengaluru, Karnataka, India.
E-mail: ashokg3s@gmail.com
Yogi Dhawane is currently a Research Associate at ICAR – Indian Institute of Horticultural Research, Bengaluru and also pursuing his Doctoral degree program from Jain University, Bengaluru, Karnataka, India.
E-mail: yogidhawane@gmail.com
Asokan Ramasamy is presently working as a Principal Scientist, at ICAR – Indian Institute of Horticultural Research, Bengaluru, Karnataka, India.
E-mail: asokan.r@icar.gov.in
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