Strategy to select the CRISPR/Cas9 target sequences of the sucrose-phosphate synthase gene for assembling an efficient gRNA spacer in shallot

Abstract

Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. Allii, is a newly identified pathogen that infects shallots. This disease can lead to yield losses ranging from 20% to 100%. The genome editing system via CRISPR/Cas-9 is a technology that can be used to modify susceptibility genes accurately and precisely. The target gene for CRISPR/Cas-9 genome editing system to develop a BLB-resistant shallot is the sucrose-phosphate synthase (SPS). One of the critical factors in the CRSPR/Cas-9 genome editing includes the preparation of single guide RNA (sgRNA) design and construction of it into an expression plasmid vector. The study aimed to develop strategies for selecting the SPS gene’s CRISPR/Cas9 target sequences to assemble an efficient gRNA spacer in shallot. The gRNA design of the SPS gene was carried out using the software at http://crispor.tefor.net/. The efficiency of sgRNAs is then filtered and predicted based on GC contents, the last four nucleotides, secondary RNA structure, and stem loops. The construction of the CRISPR/Cas9 module was carried out using the Golden Gate method. The results showed that based on the selection of target gRNA sequences in the SPS gene, one of the best gRNAs, gRNA-54/forw, has been produced for use in SPS gene editing research. The best-selected gRNA of the SPS gene was then successfully inserted into the CRISPR/Cas9 pRGEB32 cassette vector after verification using DNA sequencing analysis. Based on this result, the pRGEB32_gRNA-AcSPS construct is ready to be introduced into shallot to develop a BLB-resistant shallot variety.