Orthogonal Genome Editing and Transcriptional Activation in Tomato Breeding Using CRISPR Combo and CBE-Combo

CRISPR-Combo is an orthogonal genome engineering tool that enables simultaneous gene activation and editing in plants. This tool has previously been demonstrated for its ability to facilitate hormone free regeneration of edited lines in rice, and hence enriching genome editing events. It has also been shown to accelerate plant breeding with proof-of-concept demonstration in Arabidopsis. To broaden the applications of the CRISPR- Combo, here we showcase its application in tomato plants. By simultaneously editing the SlPSY(Phythoene Synthase)gene and activating the SlSFT(Single Flower Truss) gene , we demonstrate the functionality of the CRISPR-Combo in tomatoes and its potential to enhance plant genome editing. Firstly, the CRISPR-Combo(T-DNA 4327) and the CBE-Combo(cytosine base editor)(T-DNA 4330) induce mutation patterns that are comparable to those of the canonical Cas9 (T-DNA 4325)and Cas9n-CBE(T-DNA 4328), respectively. Secondly , through the activation of SlSFT, we highlight the potential for rapid breeding and the selection of transgene-free genome-edited plants.