CRISPR specificity in tomato

Summary

CRISPR-mediated genome editing in plants has the potential to significantly accelerate plant breeding. This technique is based on the targeted induction of double-stranded DNA breaks in the plant genome. Repair of these breaks may result in new genetic variation, which can lead to desirable traits. However, the specificity of the CRISPR-system in making DNA breaks in plants requires further investigation. Breaks at positions other than the intended target site (so called off-target sites) may result in unintended, undesirable effects. In this research, we have applied and developed several methods to study the specificity of CRISPR-Cas9 (and, to a lesser extent, CRISPR-Cas12a) in tomato in-depth. Our findings indicate that CRISPR-Cas9 and CRISPR-Cas12a rarely induce breaks at off-target sites and that off-target sites can be predicted – and thus, avoided. Additionally, this study provided valuable insight in the frequencies and types of mutations induced by CRISPR-mediated genome editing. The results presented in this thesis are useful for researchers using CRISPR-mediated genome editing as a tool, as well as for regulatory officials involved in the oversight of genome-edited crops.