Efficient nanoparticle-based CRISPR-Cas13d induced mRNA disruption of an eye pigmentation gene in the white-backed planthopper, Sogatella furcifera

Authors: MA, YUN-FENG - Guizhou University; ZHANG, MENG-QI - Guizhou University; GONG, LANG-LANG - Guizhou University; LIU, XUAN-ZHENG - Guizhou University; LONG, GUI-JUN - Guizhou University; GUO, HUAN - Guizhou University; Hull, Joe; DEWER, YOUSSEF - Agricultural Research Center Of Egypt; HE, MING - Guizhou University; HE, PENG - Guizhou University

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/7/2023
Publication Date: 12/5/2023
Citation: Ma, Y., Zhang, M., Gong, L., Liu, X., Long, G., Guo, H., Hull, J.J., Dewer, Y., He, M., He, P. 2023. Efficient nanoparticle-based CRISPR-Cas13d induced mRNA disruption of an eye pigmentation gene in the white-backed planthopper, Sogatella furcifera. Insect Science. 30(6):1552-1564. https://doi.org/10.1111/1744-7917.13203.
DOI: https://doi.org/10.1111/1744-7917.13203

Interpretive Summary: Genetic manipulation of phenotypic traits has been significantly impacted by the development of CRISPR-based technologies. While the most extensively studied and utilized CRISPR-Cas9 system functions at the level of the gene itself, Cas13 proteins that target mRNA transcripts for degradation have been reported. Members of this family of proteins, however, have only recently been recognized for their potential to impact insect pest management approaches. The utility of this system to drive targeted transcript degradation in white-backed planthoppers (common pests of rice) was assessed by injecting planthoppers with Cas13d-nanoparticle complexes specific for a gene critical in eye pigmentation. Transcripts for the target gene were significantly reduced in insects injected with the complexes relative to the controls and injected insects were characterized by bright red eyes rather than the typical olive-color of non-injected planthoppers. Taken together, the results confirm the utility of Cas13 system for mRNA disruption in insects and lay the foundation for further development of these tools in the implementation of green agricultural pest management tactics.

Technical Abstract: The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) system has driven gene manipulation technology to a new era with applications reported in organisms that span the tree of life. The utility of CRISPR-mediated editing was further expanded to mRNA following identification of the RNA-targeting Cas13 family of smaller endonuclease proteins. Application of this family to insect research, however, has been more limited. In this study, the smallest Cas13 family member, Cas13d, and guide RNAs (gRNAs) were complexed with a versatile nanomaterial (star polycation, SPc) to generate a proof-of-concept RNA-editing platform capable of disrupting mRNA expression of the eye pigmentation gene tryptophan 2,3-dioxygenase (SfTO) in white-backed planthoppers (WBPHs). The resulting red-eye phenotype was present in 19.76% (with SPc) and 22.99% (without SPc) of the treatment groups and was comparable to the red-eye phenotype generated following conventional RNA interference knockdown (22.22%). Furthermore, the Cas13/gRNA phenotype manifested more quickly than RNA interference. Consistent with the expected Cas13d mechanism, SfTO transcript levels were significantly reduced. Taken together, the results indicate that the SPc-CRISPR-Cas13d/gRNA complex negatively impacted expression of the target gene. These findings confirm the utility of this novel mRNA disruption system in insects and lay the foundation for further development of these tools in the implementation of green agricultural pest management tactics.