Designer grass pea for transgene-free minimal neurotoxin-containing seeds with CRISPR-Cas9

Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid (β-ODAP) has restricted its cultivation. Although various cultivars with low β-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal β-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the β-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the backcrossed mutant lines and they displayed no phenotypic abnormalities. Interestingly, the seed β-ODAP content ranged between 0.001 % - 0.002 % of dry weight which is 99 % lower than the wild-type. Together, our study reports the development of transgene-free, genome-edited grass peas with insignificant levels of β-ODAP in seeds for safer food in the future.