Developing an Efficient and Visible Prime Editing System to Restore Tobacco 8-Hydroxy-copalyl Diphosphate Gene for Labdane Diterpene Z -Abienol Biosynthesis

Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T 1 plants. This system was used to confer Z -abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z -abienol were detected in the leaves of homozygous T 1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z -abienol in tobacco might provide an efficient way to obtain Z -abienol in plants.