Features affecting Cas9‐induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset

SUMMARY CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single‐guide RNAs (sgRNAs) and DNA double‐strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near‐complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology‐mediated end joining (MMEJ), resembling patterns associated with high‐efficiency guides in human cells, and suggesting conserved sequence‐driven repair biases across species. In contrast, widely used human‐trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross‐species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.