Teng‐Kuei Huang, Brittney Armstrong, Patrick Schindele, Holger Puchta

Plant Biotechnology Journal;  28 January 2021

Summary

Nicotiana tabacum is a non‐food herb that has the potential to be utilized as bio‐factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre‐designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site‐specific double‐strand breaks to enhance homologous recombination‐mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas‐based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature‐insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium‐mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T‐DNA integration. Thus, we were not only able to obtain CRISPR/Cas‐based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.

See: https://onlinelibrary.wiley.com/doi/10.1111/pbi.13546

Figure 1: Mutagenesis efficiency of the SaCas9, LbCas12a, and ttLbCas12a nucleases. (a,b) The Indel efficiencies of transgenic calli after 6‐ (a) and 10‐week (b) incubation at 22°C and 28°C were analysed by TIDE, using protospacer P3. The bars represent the percentage of reads with Indels from individual calli. (c) Sequences of the protospacers used to generate DSBs at the SuRB and SuRA loci. (d) Indel analysis by next‐generation sequencing. (e) Comparison of the ratio of insertion and deletion formation after cutting by SaCas9, LbCas12a and ttLbCas12a as determined by NGS. (f) Comparison of distributions of deletion sizes obtained from LbCas12a and ttLbCas12a.