Gene Editing of Two Cyanide Synthesis-related Genes in Cassava using CRISPR/Cas9

Abstract

Cassava (Manihot esculenta) contain toxic quantities of cyanogenic glucoside. Reducing the potentially toxic cyanogenic glucoside levels in cassava could be achieved by gene editing through Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR associated protein 9 (CRISPR/Cas9), a high- precision genome editing technique in plants. Two cassava UDP- glucosyltransferase (UGT) paralogs, UGT85K4 and UGT85K5, are the genes catalyzing the synthesis of cyanogenic glucosides in plants. The study aimed to construct the CRISPR/Cas9 recombinant vector carrying single guide RNA (sgRNA) to edit UGT85K4 and UGT85K5 genes, and transform sgRNA CRISPR/Cas9 construct into KU50 cassava calli. Five sgRNAs were designed, considering the on/off target efficiency score and GC content. Those sgRNAs were ligated into pRGEB32 plasmid and introduced to competent Escherichia coli DH5a. The PCR colony and DNA sequencing were performed to verify the sgRNA insert. The amplicons showed the expected band size of recombinant vector (180 bp). Based on sequencing results, only one sgRNA was successfully ligated into the plasmid that will be used to edit the UGT85K5 gene. The sgRNA CRISPR/Cas9 construct in pRGEB32 plasmid was successfully transformed into Agrobacterium tumefaciens and introduced into KU50 cassava calli through co-cultivation. The overgrowth of Agrobacterium occurred after the co-cultivation phase.