Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants

Background TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C center dot G-to-T center dot A editing. This technology has not been optimized for use in plant cells.Results To systematically investigate TALE-DdCBE architectures and editing rules, we established a beta-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C center dot G-to-T center dot A conversions in their chloroplast genome, as well as N. benthamiana with C center dot G-to-T center dot A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants.Conclusions Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.