Journal of Environmental Biology

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

S. Ramesh and A. Ghosh*     

Insect Vector Laboratory, Advanced Centre for Plant Virology, ICAR-Indian Agricultural Research Institute, New Delhi-110 012, India

Received: 17 June 2025                   Revised: 14 October 2025                   Accepted: 24 December 2025

*Corresponding Author Email : amal4ento@gmail.com                        *ORCiD: https://orcid.org/0000-0001-6634-5771

Abstract

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 RNP complexes 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 RNP combinations 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

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