DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

Despite their well-defined genetic background, repeat expansion diseases (REDs) still represent an unmet medical need, with no causative therapy offered to patients. The strategy of repeat shortening using genome editing tools is very attractive because a single intervention can result in permanent repair of the disease-causing mutation. However, a limited understanding of DNA repair mechanisms in repetitive sequences complicates the prediction and control of the editing effects. Using a CRISPR interference (CRISPRi) screen, we identified pathways and factors responsible for the repair of staggered cuts generated by Cas12a within CAG repeat tracts. This analysis revealed a central role for interstrand crosslink (ICL) repair factors in mediating CAG repeat contraction, with DCLRE1A emerging as a key effector. We demonstrated that DCLRE1A recognizes and binds structures generated by Cas12a. Moreover, DCLRE1A interacts with SLX4, promoting the generation of pure contractions, and with POLI, leading to the formation of inverted repeats at the break site during template switching mechanisms. We then used this knowledge to increase the contribution of pure contractions to the pool of editing outcomes using fusions of Cas12a with DNA repair proteins. Our study indicates that Cas12a can be used as an effective tool for generating repeat contractions. Although the mechanisms leading to repeat shortening are complex, understanding them can help researchers develop more precise therapeutic strategies with greater control of the editing process.