In vitro evaluation of sgRNA-Cas9 RNPs for knocking out essential pigmentation genes in Bemisia tabaci

Aim: This study aimed to design and screen highly effective sgRNAs for RNP-based genome editing in Bemisia tabaci Asia II 1and to validate the efficiency and specificity of the synthesized RNPcomplexes through in-vitro assays, thereby enabling precise and targeted gene editing. Methodology: Amplicons of two essential pigmentation-related genes were used to evaluate the efficiency of the designed sgRNA-Cas9 complex through an in-vitro cleavage assay, enabling the identification of optimal RNPcombinations for in-vivo targeted gene editing. Results: The LAC2 and w genes of B. tabaci were successfully characterized, and submitted to NCBI (accession numbers PV012220 and PV012221). In-vitro cleavage assays confirmed efficient and precise cutting of target gene amplicons by specific Cas9-sgRNA RNP complexes at expected positions. Interpretation: Phylogenetic analysis revealed that the LAC2 and w genes were highly conserved in B. tabaci. In-vitro cleavage with sgRNA-Cas9 RNP complexes provides a rapid and effective pre-validation of CRISPR-Cas functionality in insects. This approach eliminates the need for preliminary cell line testing before in-vivo application. Key words:Gene editing, LAC2,Ribonucleoprotein complex, w gene, Whitefly