Abstract:
Cassava is the world’s most essential food root crop, generating calories to
millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots
contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of
residual cyanogens results in cyanide poisoning due to conversion of the
cyanogens to cyanide in the body. There is a need for acyanogenic cassava
cultivars in order for it to become a consistently safe and acceptable food, and
commercial crop. In recent years, the CRISPR/Cas system, has proven to be the
most effective and successful genome editing tool for gene function studies
and crop improvement. In this study, we performed targeted mutagenesis of
the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacteriummediated transformation. The vector design resulted in knockout in
cotyledon-stage somatic embryos regenerated under hygromycin selection.
Eight plants were recovered and genotyped. DNA sequencing analysis revealed
that the tested putative transgenic plants carried mutations within the
MeCYP79D1 locus, with deletions and substitutions being reported upstream
and downstream of the PAM sequence, respectively. The levels of linamarin and
evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to
seven-fold. Nevertheless, the cassava linamarin and cyanide were not
completely eliminated by the MeCYP79D1 knockout. Our results indicate that
CRISPR/Cas9-mediated mutagenesi s is as an alternative approach for
development of cassava plants with lowered cyanide content.
