Journal: bioRxiv

Article Title: DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

doi: 10.1101/2025.11.18.689026

Figure Lengend Snippet: DCLRE1A recognizes and processes structures arising from 5’ overhangs. A. DCLRE1A accumulates at Cas12a-induced DNA breaks within a repetitive target. Representative images show the nuclei of control RPE-1 (without DSB induction) (Control) and Cas12_g14-treated RPE-1 cells infected with multiple viruses carrying 40 CAG repeats. Scale bar = 10 μm. The graph shows that the percentage colocalization coefficient increased from 13.32% to 32.91% for DCLRE1A–γH2AX colocalization following DSB induction (n = 14 and 13, respectively). For each cell, 10 to 16 micrographs were captured along the Z-axis, from which 3 micrographs were selected for calculations. Statistical analysis was conducted using the Mann‒Whitney and Kolmogorov‒Smirnov tests. Asterisks indicate levels of statistical significance: **** p ≤ 0.0001. B. DCLRE1A recognizes and binds to 5’ overhangs in human cells. Schematic representation of the coimmunoprecipitation experiment. DCLRE1A bound to the annealed oligonucleotides that mimic the Cas12a-generated 5’ overhangs. HEK293T_35CAG_ATN1 cells were transfected with plasmids expressing either DCLRE1A or the nickase Cas9 (nCas9) with a gRNA targeting the fragment of the HTT gene upstream of the CAG repeat tract (negative control). Cells were collected at 0 h, 4 h, 12 h, and 24 h following oligonucleotide delivery. C. The purified recombinant DCLRE1A protein exhibited endo- and exonucleolytic activities toward structures formed by 5’ overhangs in vitro. The DCLRE1A protein (25, 50, and 100 nM) was added to the annealed oligonucleotides (enOligo and exoOligo) and a positive control with a 5’ overhang. The digested products were separated on a PAGE gel under denaturing conditions. The structures arising from annealed oligonucleotides are shown below the denaturing gel. Asterisks indicate 5′-FAM-labeled nucleotides. P denotes a phosphorothioate substitution that prevents exonuclease cleavage.

Article Snippet: Cells were collected 48 h after g14 delivery, and DNA was isolated using a Genomic DNA Isolation Kit (Norgen, Biotek Corp.) according to the manufacturer’s instructions.

Techniques: Control, Infection, Generated, Transfection, Expressing, Negative Control, Purification, Recombinant, In Vitro, Positive Control, Labeling

Journal: bioRxiv

Article Title: DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

doi: 10.1101/2025.11.18.689026

Figure Lengend Snippet: SLX4 and DCLRE1A influence the occurrence of pure contraction events following Cas12a-induced DNA breaks. A. The graph shows DNA-related genes whose knockdown resulted in a drastic decrease in the number of events with 2 and 3 CAG repeats. B. The graph shows the percentage of pure contractions following the knockdown of DCLRE1A (DCLRE1A), SLX4 (SLX4), and both DCLRE1A and SLX4 (DCLRE1A + SLX4). The bars in the graph represent the mean values of the pure contracted variants ± SEMs. The p-values are indicated by asterisks (*p < 0.05 and **p < 0.01). This experiment was repeated twice. C. Volcano plot showing DNA repair-related genes detected in the BioID analysis whose expression decreased or was enriched after Cas12a-induced DNA breaks within CAG repeats. Here, we compared cells expressing Cas12a (Cas12a-BirA_g14) and cells expressing dCas12a (dCas12a-BirA_g14), both of which were treated with g14. D. Comparison of the cleavage effect(Cas12a_BirA_g14 vs. dCas12a_BirA_g14), and binding effect (dCas12a_BirA_g14 vs. dCas12a_BirA) for only the DNA-related proteins identified in the BioID analysis.

Article Snippet: Cells were collected 48 h after g14 delivery, and DNA was isolated using a Genomic DNA Isolation Kit (Norgen, Biotek Corp.) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Comparison, Binding Assay

Journal: bioRxiv

Article Title: DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

doi: 10.1101/2025.11.18.689026

Figure Lengend Snippet: Fusion proteins increase the number of pure contractions. A. Schematic representation of the vector expressing the Cas12a-DNA repair factor fusion protein. B. NGS data revealed variations in the contribution of different types of events mediated by the Cas12a-DNA repair factor fusion protein in HEK293T_35CAG_ATN1 cells. BIRA-treated cells served as controls. C. Timeline of the experiment for determining the frequency of pure contraction events after Cas12a-DNA repair factor treatment in HEK-Flp-In_T-Rex_64CAG cells. Representative flow cytometry plots are shown below. The first plot displays untreated cells, whereas the second shows cells treated with doxycycline. The third and fourth plots show cells treated with plasmids expressing the fusion protein and BFP and cells treated with plasmids and g14, respectively. D. The graph shows the percentage of GFP-positive cells (pure contraction variants) following treatment with the fusion proteins. The statistical analysis was performed using an unpaired t-test (n = 3), with a p-value of ≤ 0.0001.

Article Snippet: Cells were collected 48 h after g14 delivery, and DNA was isolated using a Genomic DNA Isolation Kit (Norgen, Biotek Corp.) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Expressing, Flow Cytometry