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Results for “MMEJ”

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4 recordsLinked to original sources
PubMed2025

Disruption of Microhomology-mediated End-joining in Ewing Sarcoma.

Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POLΘ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POLΘ expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POLΘ mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS.

Alternative end-joining↗
PubMed2026

Mitotic microhomology-mediated break-induced replication promotes chromoanasynthesis.

Chromoanasynthesis is a form of complex chromosomal rearrangement (CCR) commonly detected in cancers and congenital disorders, but the mechanism underlying its generation remain elusive. Here we develop a single-molecule long-read DNA sequencing approach to characterise ultra-complex mutational events, consistent with chromoanasynthesis, occurring at shortened telomeres and sub-telomeric DNA double-strand breaks in human cells. Our data reveal that chromoanasynthesis is generated by microhomology-mediated break-induced replication (MM-BIR), occurring specifically in mitosis. Surprisingly, this mitotic pathway involves a collaboration between microhomology-mediated end-joining (MMEJ) and BIR, where MMEJ proteins initiate a Polδ-dependent BIR pathway that is regulated by PIF1, POLD3 and PCNA. This pathway is highly prone to template switching and can generate dramatic amplification of genomic loci in a single event. Our findings help explain the extreme mutagenic nature of chromoanasynthesis and establish mitotic MM-BIR as a key driver of CCRs, with important implications for the origin of cancers and congenital disorders.

Humans↗
PubMed2026

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

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.

Solanum lycopersicum↗
PubMed2026

Interfering with DNA repair pathways to enhance CRISPR-Cas9-mediated homology-directed repair in a chelicerate genetic model.

The two-spotted spider mite, Tetranychus urticae, is a major pest and an emerging genetic model. Recent CRISPR-Cas9 advances, especially the SYNCAS method for maternal delivery of Cas9 ribonucleoproteins, have enabled precise genome editing in this and other difficult-to-transform arthropods. Yet SYNCAS-mediated knockins vary in efficiency, possibly due to competition between DNA repair pathways, whose mechanisms in T. urticae and other chelicerates remain unknown. Here, we provide the first functional analysis of double-strand break repair in a chelicerate. Loss of DNA polymerase theta (Polθ) redirects repair almost entirely toward homology-directed repair, whereas absence of Ligase IV has no detectable impact. Using a reporter assay targeting phytoene desaturase, we demonstrate that Polθ-deficient strains enhance incorporation of repair templates, even when mutations are distant from the cut site. Also, insertion of larger fragments is improved. Finally, disrupting Polθ imposes only a modest fitness cost, highlighting its value for future genome engineering in this species.

Acari↗