CRISPR/Cas9 Protection System for Chilli Leaf Curl Virus
Rakesh Kumar Verma, Mansi Mishra, Avinash Marwal, Afia Akhtar, N. Petrov, Rajarshi Kumar Gaur
Abstract
Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) targeted genome editing system has already modernized plant science research and has been used to provide resistance sources against plant viral diseases. In this study, we have used CRISPR/Cas9 technology to introduce sequence-specific targeted gene silencing in Nicotiana benthamiana to successfully engineer complete resistance to Chilli leaf curl virus (ChiLCuV). We proposed the multiplex type sgRNA (short guide) with CRISPR/Cas9 to increase the effective control against IR and Rep gene of DNA-A of ChiLCuV. Highly conserved regions having PAM (Protospacer Adjacent Motif) sequence were selected to design sgRNA cassette and inserted into binary vectors. This cassette was used to transform N. benthamiana by the leaf disc method and further transgenic plants were challenged with ChiLCuV and screened for resistance. Our results suggest that the resistance shown in N. benthamiana against ChiLCuV is quite stable.