Design and validation of CRISPR/Cas9 sgRNAs and vector construction for targeting the LsBOS gene in grasspea (Lathyrus sativus L.)

Abstract

Grasspea is an important grain legume crop due to its hardiness and high protein content. However, the presence of a neurotoxic compound β -ODAP, which is associated with lathyrism, limits its widespread cultivation and consumption. In this study, the LsBOS gene, which encodes β -ODAP synthase, was PCR-amplified from genomic DNA of grasspea cv. Mahateora and sequenced. Sequence analysis confirmed that the LsBOS gene is intronless. Multiple sequence alignment identified 13 single nucleotide polymorphisms (SNPs) within the coding region of the gene; however, none were predicted to affect protein function. No sequence variation was detected within the conserved HXXXD domain region of the gene. Three single-guide RNAs (sgRNAs) targeting the conserved region were designed and evaluated using an in vitro cleavage assay. The assay confirmed that all three sgRNAs specifically recognized and cleaved their intended target sequences, supporting their suitability for CRISPR/Cas9 applications. The validated sgRNAs were subsequently cloned into the CRISPR/Cas9 vector pKSE401 to generate genome-editing constructs targeting the LsBOS gene in grasspea. The developed constructs and experimentally validated sgRNAs provide foundational resources for future genome-editing studies aimed at reducing β -ODAP content in grasspea. Genome editing in grasspea using these constructs was not evaluated in the present study.