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MMEJ induced by Cas9 and Cas9n exhibits distinct genetic dependence. ( A-C ) MMEJ was assayed in U2OS (EGFP-MMEJ) cells expressing shRNAs for Polθ ( A ), LIG3 ( A ), RPA2 ( B <t>),</t> <t>MRE11</t> ( C ) or <t>CtIP</t> ( C ) after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( D ) MMEJ was assayed in WT or POLQ -KO mES (EGFP-MMEJ) cells four days after transfection of plasmids encoding g2/Cas9 WT or g2/Cas9 D10A . ( E ) mES (EGFP-MMEJ) cells with POLQ WT allele or knock-in (KI) alleles containing mutations of K120G or D2494P/E2495R were assayed for MMEJ after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( F ) In vitro biochemical assay showing Polθ-HelD activity in unwinding dsDNA to facilitate strand exchange with ssDNA carrying 15 bp homology. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrate and incubated with Polθ-HelD, ATP and/or RPA, and the reaction products were resolved on a non-denaturing gel. ( G ) In vitro biochemical assay showing strand displacement DNA synthesis by Polθ-HelD and Polθ-PolD. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrates and incubated with Polθ-HelD, ATP and/or RPA, followed by DNA extension with Polθ-PolD with 0.1 mM dNTPs, and the reaction products were resolved on a denaturing gel.
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MMEJ induced by Cas9 and Cas9n exhibits distinct genetic dependence. ( A-C ) MMEJ was assayed in U2OS (EGFP-MMEJ) cells expressing shRNAs for Polθ ( A ), LIG3 ( A ), RPA2 ( B ), MRE11 ( C ) or CtIP ( C ) after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( D ) MMEJ was assayed in WT or POLQ -KO mES (EGFP-MMEJ) cells four days after transfection of plasmids encoding g2/Cas9 WT or g2/Cas9 D10A . ( E ) mES (EGFP-MMEJ) cells with POLQ WT allele or knock-in (KI) alleles containing mutations of K120G or D2494P/E2495R were assayed for MMEJ after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( F ) In vitro biochemical assay showing Polθ-HelD activity in unwinding dsDNA to facilitate strand exchange with ssDNA carrying 15 bp homology. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrate and incubated with Polθ-HelD, ATP and/or RPA, and the reaction products were resolved on a non-denaturing gel. ( G ) In vitro biochemical assay showing strand displacement DNA synthesis by Polθ-HelD and Polθ-PolD. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrates and incubated with Polθ-HelD, ATP and/or RPA, followed by DNA extension with Polθ-PolD with 0.1 mM dNTPs, and the reaction products were resolved on a denaturing gel.

Journal: bioRxiv

Article Title: Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

doi: 10.64898/2026.01.15.699632

Figure Lengend Snippet: MMEJ induced by Cas9 and Cas9n exhibits distinct genetic dependence. ( A-C ) MMEJ was assayed in U2OS (EGFP-MMEJ) cells expressing shRNAs for Polθ ( A ), LIG3 ( A ), RPA2 ( B ), MRE11 ( C ) or CtIP ( C ) after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( D ) MMEJ was assayed in WT or POLQ -KO mES (EGFP-MMEJ) cells four days after transfection of plasmids encoding g2/Cas9 WT or g2/Cas9 D10A . ( E ) mES (EGFP-MMEJ) cells with POLQ WT allele or knock-in (KI) alleles containing mutations of K120G or D2494P/E2495R were assayed for MMEJ after g2/Cas9 WT or g2/Cas9 D10A cleavage. ( F ) In vitro biochemical assay showing Polθ-HelD activity in unwinding dsDNA to facilitate strand exchange with ssDNA carrying 15 bp homology. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrate and incubated with Polθ-HelD, ATP and/or RPA, and the reaction products were resolved on a non-denaturing gel. ( G ) In vitro biochemical assay showing strand displacement DNA synthesis by Polθ-HelD and Polθ-PolD. 32 P-5’-labeled ssDNA was mixed with the indicated dsDNA substrates and incubated with Polθ-HelD, ATP and/or RPA, followed by DNA extension with Polθ-PolD with 0.1 mM dNTPs, and the reaction products were resolved on a denaturing gel.

Article Snippet: Antibodies used in immunoblotting are: MRE11 (Cell Signaling Technology, 4895), CtIP (Proteintech, 12624-1-AP), RPA1 (Sigma-Aldrich, NA13), RPA2 (Bethyl, A300-244A), PCNA (Cell Signaling Technology, 13110), MCM2 (Proteintech, 10513-1-AP), DNA2 (Proteintech, 18727-1-AP), KU70 (Santa Cruz Biotechnology, sc-17789), HA (Santa Cruz Biotechnology, sc-7392), Flag (Sigma-Aldrich, F1804), BLM (Bethyl, A300-110A), EXO1 (Bethyl, A302-640A), ATR (Santa Cruz Biotechnology, sc-515173), BRCA1 (Santa Cruz Biotechnology, sc-6954), RAD51 (Santa Cruz Biotechnology, sc-398587), LIG3 (Proteintech, 26583-1-AP), γH2AX (Upstate, 07-164), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, 115-035-146), and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, 111-035-144).

Techniques: Expressing, Transfection, Knock-In, In Vitro, Activity Assay, Labeling, Incubation, DNA Synthesis

Fork-MMEJ functions together with BIR to repair seDSBs on broken forks. ( A ) Schematic drawings of the EGFP-MMEJ/mCherry-BIR reporter and the repair products by cMMEJ and BIR after Cas9 WT cleavage. The gRNA2 cleavage site is indicated. ( B ) MMEJ or BIR was scored in U2OS (EGFP-MMEJ/mCherry-BIR) cells by FACS to determine EGFP or mCherry positive cells after cleavage by gRNA2 with Cas9 WT and Cas9 D10A . ( C ) Time course experiments were performed in U2OS (EGFP-MMEJ/mCherry-BIR) cells to analyze MMEJ and BIR after cleavage by gRNA2 with Cas9 WT and Cas9 D10A . ( D - F ) MMEJ and BIR was assayed in U2OS (EGFP-MMEJ/mCherry-BIR) cells expressing shRNAs for CtIP ( D ), MRE11 ( D ), PIF1( E ) or Polθ ( F ) after expressing gRNA2 with Cas9 and Cas9n.

Journal: bioRxiv

Article Title: Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

doi: 10.64898/2026.01.15.699632

Figure Lengend Snippet: Fork-MMEJ functions together with BIR to repair seDSBs on broken forks. ( A ) Schematic drawings of the EGFP-MMEJ/mCherry-BIR reporter and the repair products by cMMEJ and BIR after Cas9 WT cleavage. The gRNA2 cleavage site is indicated. ( B ) MMEJ or BIR was scored in U2OS (EGFP-MMEJ/mCherry-BIR) cells by FACS to determine EGFP or mCherry positive cells after cleavage by gRNA2 with Cas9 WT and Cas9 D10A . ( C ) Time course experiments were performed in U2OS (EGFP-MMEJ/mCherry-BIR) cells to analyze MMEJ and BIR after cleavage by gRNA2 with Cas9 WT and Cas9 D10A . ( D - F ) MMEJ and BIR was assayed in U2OS (EGFP-MMEJ/mCherry-BIR) cells expressing shRNAs for CtIP ( D ), MRE11 ( D ), PIF1( E ) or Polθ ( F ) after expressing gRNA2 with Cas9 and Cas9n.

Article Snippet: Antibodies used in immunoblotting are: MRE11 (Cell Signaling Technology, 4895), CtIP (Proteintech, 12624-1-AP), RPA1 (Sigma-Aldrich, NA13), RPA2 (Bethyl, A300-244A), PCNA (Cell Signaling Technology, 13110), MCM2 (Proteintech, 10513-1-AP), DNA2 (Proteintech, 18727-1-AP), KU70 (Santa Cruz Biotechnology, sc-17789), HA (Santa Cruz Biotechnology, sc-7392), Flag (Sigma-Aldrich, F1804), BLM (Bethyl, A300-110A), EXO1 (Bethyl, A302-640A), ATR (Santa Cruz Biotechnology, sc-515173), BRCA1 (Santa Cruz Biotechnology, sc-6954), RAD51 (Santa Cruz Biotechnology, sc-398587), LIG3 (Proteintech, 26583-1-AP), γH2AX (Upstate, 07-164), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, 115-035-146), and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, 111-035-144).

Techniques: Expressing