In a bid to discover how to maximize the tobacco plant's potentials as bio-factories, an international team of scientists investigated which CRISPR technology was more efficient in incorporating two herbicide resistance modifications simultaneously in the tobacco' SuRB gene. The results not only exhibited which CRISPR technology was more efficient, but that it was also possible to inherit gene-targeted modifications.

Tobacco (Nicotiana tabacum) is a plant often considered for use as a bio-factory in generating medicines and vaccines, while the CRISPR-Cas nuclease technology allows the induction of site-specific double-strand breaks for better homologous recombination-mediated gene targeting. Considering that gene targeting technology continues to be enhanced by replacing SpCas9 with other CRISPR-Cas-based nucleases, the scientists decided to test SaCas9 and a temperature-insensitive ttLbCas12a using tobacco plant to target the SuRB gene using an optimized protocol for Agrobacterium-mediated tobacco transformation and tissue culture.

The scientists found that ttLbCas12a outperformed SaCas9 in terms of gene targeting efficiencies by up to a third of the inoculated cotyledons. They recorded that the conversion tract length of the gene targeting reaction can stretch to over 250 bp long, and they were also able to obtain multiple heritable heterozygous and non-T-DNA integrated biallelic gene target events in tobacco. This is the first time that researchers are able to obtain CRISPR-Cas-based heritable gene-targeted events in tobacco.

Read the full methodology in Plant Biotechnology Journal.