In silico design of CRISPR/Cas9 guide RNA for the knockout of the phytoene desaturase gene in sweet potato (Ipomoea batatas L.)

Abstract

This study aimed to design in silico guide RNA (sgRNA) for CRISPR/Cas9-mediated knockout of the phytoene desaturase (PDS) gene in sweet potato (Ipomoea batatas L.). The sequence of the coding region of the IbPDS gene is 1791 base pairs (bp) long, and these, in turn, are equivalent to 572 amino acids. The amino acid sequence of the IbPDS gene was compared with the homologous sequences of other nearby plant species, showing that it presents a close similarity with PDS of Ipomoea triloba and Ipomoea nil with 98.60% and 97.73%, respectively. CRISPR RGEN Tools provided 113 results for the IbPDS gene, filtering to 24 and selecting three sgRNA sequences for the design of the gene editing vector, which were sgRNA 1 (5'-ACCTCATCAGTCACCCTGTCNGG-3'), sgRNA 2 (5'- CCTCCAGCAGCAGTATTGGTTGGTTTGNGG -3') and sgRNA 3 (5'- CTGAACTCTCCTGGTTGGTTGTTNGG -3'). The predicted secondary structures of the selected sgRNAs present efficient sgRNA structures for gene editing of the target gene. The PMH-Cas9- 3xsgRNA vector for CRISPR/Cas9-mediated knockout of the IbPDS gene was designed in silico with three sgRNA sequences and one Hygromycin resistance marker. Keywords: Gene editing, sgRNA, IbPDS, gene editing vector, Hygromycin.