DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

Dabrowska, M.; Siegner, S.; Misiukiewicz, A.; Bandaruk, Y.; Schroeder, M. S.; Muhar, M.; Kreutzer, S.; Corn, J. E.; Olejniczak, M.

doi:10.1101/2025.11.18.689026

bioRxiv · 10.1101/2025.11.18.689026

DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

Dabrowska, M.·Siegner, S.·Misiukiewicz, A.·Bandaruk, Y.·Schroeder, M. S.·Muhar, M.·Kreutzer, S.·Corn, J. E.·Olejniczak, M.

View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

cell biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Dabrowska, M., Siegner, S., Misiukiewicz, A., Bandaruk, Y., Schroeder, M. S., Muhar, M., Kreutzer, S., Corn, J. E., Olejniczak, M.. 2025-11-18. DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks. https://doi.org/10.1101/2025.11.18.689026

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗

bioRxiv2026

High-content screening identifies FDA-approved activators of mitophagy

Mitochondrial diseases are highly heterogeneous disorders caused by defects in oxidative phosphorylation, for which only limited therapeutic options are available. The elimination of dysfunctional mitochondria via mitophagy activation is an attractive strategy to treat several mitochondrial diseases, irrespective of the underlying genetic defect. To identify pharmacological activators of this pathway, we performed a high-content screening of 1,971 FDA-approved compounds using cellular reporters of TFEB nuclear translocation and mitophagy. This screening identified 294 compounds that promoted TFEB translocation, nine of which increased the number of mitolysosomes without compromising cell viability or damaging mitochondria. Among the selected compounds, Meptazinol and Ponatinib emerged as the top-ranked mitophagy inducers in fibroblasts from patients with complex I and IV deficiency, respectively, and were prioritised for further characterisation. However, they did not rescue the bioenergetic defect, suggesting that the stimulation of mitophagy per se is not sufficient to ameliorate the OXPHOS defect in mitochondrial diseases. Overall, our findings establish a scalable platform for the identification of pharmacological activators of TFEB and identify candidate compounds for therapeutic modulation of mitophagy.

cell biology↗

bioRxiv2026

Matrix Gla Protein deficiency reveals a previously unrecognized mucosal calcification in the mouse airways

Tracheal calcification is a common finding that is considered to have limited clinical significance when detected most often in elderly individuals. Despite its frequent occurrence, the cellular and molecular mechanisms at the origin of tracheal calcification remain largely unexplored. In this context, we aimed to characterize the development of tracheal calcification, and to determine the role of matrix Gla protein (MGP), a major inhibitor of ectopic calcification whose genetic or pharmacologic deficiency is associated with premature tracheal calcification in humans. We found that wild-type mice undergo an unexpected early physiologic calcification of the tracheal cartilage rings beginning around one month after birth. This calcification progresses in a dynamic rostro-caudal sequence through a process involving terminal differentiation of tracheal chondrocytes. These findings challenge the prevailing view that tracheal cartilage is a permanent hyaline cartilage and instead reveals an early and highly organized physiologic calcification process. Furthermore, our findings further identify MGP as a key regulator of calcification within the respiratory tract. Indeed, MGP deficiency not only markedly accelerates tracheal cartilage calcification through a similar pattern and mechanism but also induces a previously unrecognized calcification of the tracheal mucosa, a lesion absent in wild-type mice. These alterations could partly explain the respiratory manifestations observed in MGP-deficient patients having Keutel syndrome, which include dyspnea, cough, stridor, and recurrent infections, symptoms previously being mainly attributed to calcification of the tracheal cartilage rings.

cell biology↗