Designed gRNAs for CRISPR-Cas9 based antifungal resistance in eggplant.

Abstract

Eggplant is an important vegetable crop and is a good source of antioxidants, minerals, and vitamins. It has been used in ancient medicines for the treatment of multiple diseases. However, the cultivated varieties of eggplant are susceptible to numerous pathogens and pests that have a negative impact on vegetable crops. Increased resistance achieved through resistance genes (R genes) is limited in eggplant breeding due to the fact that R genes are typically specific to a pathogen race and can be quickly surpassed by pathogen evolution. The susceptibility genes (S genes) in plants facilitate pathogen entry and proliferation, thus disabling these genes might be beneficial for providing a broad range and durable resistance against pathogens. Reports on crops such as Arabidopsis, rice, wheat, citrus, and tomatoes have highlighted that the knockout mutants of the S genes are tolerant to multiple different pathogens. The CRISPR/Cas9 system facilitates plant genome editing that can be utilized efficiently for crop improvement. In the current work, we have identified the homologs of candidate S genes DMR1, DMR6, EDR1, and PMR4/5/6 in the eggplant genome and designed and screened putative gRNAs against the identified target loci. The gRNAs were screened and selected on the basis of recognition of the PAM sequence, the MIT score, their minimum free energy, and the secondary structure. Five gRNAs for each gene homolog were selected after an in-depth analysis of all the predicted gRNAs using the above-mentioned criterion.