Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a Teng‐Kuei Huang, Brittney Armstrong, Patrick Schindele,Holger Puchta* Botanical Institute II|Karlsruhe Institute of Technology, Karlsruhe, Germany 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.