Fig. 5: SSA contributes to repair of DSBs in endogenous genomic loci. | Nature Communications

Fig. 5: SSA contributes to repair of DSBs in endogenous genomic loci.

From: Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks

Fig. 5: SSA contributes to repair of DSBs in endogenous genomic loci.The alternative text for this image may have been generated using AI.

a Schematic representation of SSA repair of a Cas9-induced DSB at the Alu_1 target site in the FANCA gene. Indicated are the primers (in red) to amplify the SSA repair product, and the nucleases that perform end-resection (orange pacman) or ssDNA flap removal (yellow pacman). b Table indicating the subfamily, chromosomal location, and level of sequence homology of the Alu elements that could anneal during SSA repair of the DSB at the Alu_1 target loci. c, d HEK 293T DSB-Spectrum_V2 cells (c) or regular HEK 293T cells (d) were transfected with a nontargeting control siRNA (“-” lanes) or Rad52-targeting siRNA, then retransfected with the indicated Cas9-sgRNA constructs. Genomic DNA was isolated and SSA DSB-repair products PCR amplified. Representative gel images are shown (n = 2). Expected size of the SSA repair product and the quantified SSA band intensities are shown at the top and the bottom of the image, respectively. e Western blot of lysates from panels c and d. Red arrow indicates Rad52, asterisk indicates nonspecific band. f, g As in panels c and d, but excluding siRNA transfection, and including NU7441 treatment (2 μM). In the FANCA panel, arrows indicate the SSA products between the Alu_1 element and either the Alu_2 element (red), or a more downstream AluSx element (black). Representative gel images are shown (n = 2). h Schematic showing the genomic location and level of homology between the HBB and HBD genes. Zoom shows DNA and amino acid sequence at the start of the HBB gene (coding strand). Indicated are the sequence recognized by the HBB-targeting sgRNA, and the sickle cell disease-causing A > T mutation (bold red). i HEK 293T cells were transfected with Cas9 and either a control sgRNA or an HBB-targeting sgRNA. Cells were treated with NU7441 (2 μM) or vehicle control and the SSA DSB-repair products were PCR amplified from genomic DNA. The expected size of the SSA repair product is shown at the top, quantification of SSA band intensities is shown at the bottom.

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