Expression and Purification of Cas12 (Lbcpf1) and Its Application in Detection of Tomato Leaf Curl New Delhi Virus-potato

Abstract

Tomato Leaf Curl New Delhi Virus (ToLCNDV) was found to infect tomato and other members of Solanaceous crops. But lately it has been reported to infect a range of plant species at various geographical locations. There are many reports indicating spread of ToLCNDV to other vegetable and fiber crops, inflicting severe economic losses. Symptoms of ToLCNDV on different host plants include yellow mosaic, leaf curling, vein swelling and plant stunting, which overlap with many other viruses or disease conditions and thus detection is not possible on the basis of symptoms. So, it becomes very essential to develop fast, specific and efficient diagnosis techniques for detection of such a devastating and rapidly spreading plant virus. Conventional approaches such as antigen-antibody interaction, AFLP and PCR, etc. having some limitations could be overcome by ‘CRISPR’ based detection of targeted nucleic acids. CRISPR renowned for its precise and sensitivity as targeted genome editing (cis-cleavage) tool. Prior to the recognition of targeted genome by CRISPR-Cas complex, its ‘Collateral activity’ (trans-cleavage) gets activated. Which could be utilised as attomolar level sensitive, novel nucleic acid detection platform with the help of random ssDNA (reporter molecules) labelled at one end with fluorophore and other with quencher. Such an approach will be more sensitive, specific, faster and efficient to perform compared to available diagnostic methods. In this study, we’re interested in designing of specific gRNAs (in silico) against ToLCNDV-Potato alongside with expression & purification of Cas12a (LbCpf1) through IMAC technique then check their efficacy in CRISPR based diagnosis of ToLCNDV from potato samples.