Key determinants of CRISPR/Cas9 induced inversions in tomato

This project explores the factors influencing the induction of chromosomal inversions ranging from hundreds to millions of base pairs in plants using a novel CRISPR/Cas9-based method. By employing a standardized transduction normalisation approach with a reference guide RNA (gRNA) for consistent cleavage and a variable gRNA to manipulate break distances, we examined how mutation frequencies correlate with inversion sizes. Our findings indicate that inducing large genomic inversions (over 1 Mb) is significantly less frequent, suggesting a possible limitation in the plant-specific variant of the canonical non-homologous end-joining (c-NHEJ) repair mechanism, which may involve the active transport of large chromosomal segments to dedicated repair sites. This study contributes to our understanding of genomic engineering limits and mechanisms in plants, providing insights that could enhance genetic editing techniques.