A novel needleless delivery of Cas9 ribonucleoprotein complex in Bemisia tabaci embryos for gene editing.

The CRISPR/Cas9 system, particularly the Cas9-sgRNA ribonucleoprotein (RNP) complex, offers a highly efficient platform for gene editing. However, traditional microinjection methods for RNP delivery in insect embryos are labor-intensive, technically demanding, and often reduce embryo viability, especially in species with fragile, microscopic embryos. In this study, a novel, non-invasive delivery method for the RNP complex in tiny insect embryos has been optimized. Whitefly, Bemisia tabaci, an invasive insect pest of agricultural importance and vector of plant diseases, was considered as a model organism. A needleless dechorionation-permeabilization method was employed to deliver the RNP complex to the early embryonic stage of B. tabaci embryos. B. tabaci heat shock protein 70 (hsp70) gene, which interacts with the begomovirus coat protein, aiding in its internalization and successful transmission by B. tabaci in a persistent circulative manner, was targeted for knockout. Two sgRNAs were synthesized via in vitro transcription, and the Cas9-sgRNA complexes were validated by in vitro cleavage assays. The localization of the GFP-labelled Cas9-sgRNA complex confirmed successful RNP delivery, as observed through confocal microscopy. A survival rate of 18% of the embryos was recorded post-RNP delivery. Sequencing of treated embryos showed 25- and 28-nucleotide deletions in the hsp70 exon. Synthego ICE analysis revealed up to 84% gene knockout efficiency. This method enables batch processing of embryos, drastically reducing delivery time and associated costs while improving throughput. Hsp70 KO B. tabaci mutants generated in the study are expected to be incompetent begomovirus transmitters, which would help restrict the spread of the virus. Our study overcomes a key bottleneck in CRISPR/Cas delivery to small insect embryos, opening new avenues for rapid, high-throughput, and cost-effective RNP delivery methods in insect embryos. The novel non-invasive methods would be helpful in the deployment of gene editing for sustainable pest control.