Fig. 1: CST-complex promotes tandem duplication formation at DSBs with 3′ ssDNA overhangs.

a Schematic representation of the generated HPRT-eGFP gene and the two main target sites used in this study (exon 3 and eGFP, see Supplementary Fig. 1 for details). b FACS-plots showing the number of GFP-positive and GFP-negative cells in untransfected wild-type HPRT-eGFP cells and in HPRT-eGFP cells transfected with Cas9-N863A and two sgRNAs targeting exon 3 or eGFP seven days post-transfection. c Graphic illustration for tandem duplication formation at DNA-breaks with complementary 3′ ssDNA overhangs. d Relative HPRT-eGFP mutation frequency for the indicated mES cell lines (using two independent clones per genotype) transfected with Cas9‐N863A and sgRNAs targeting eGFP, producing DSBs with 3′ ssDNA overhangs. The data shown represent the mean ± SEM (n = 4) and are expressed as a fraction of the mutation frequency observed in wild‐type cells (set to 1). Statistical significance was calculated via a two-tailed unpaired t‐test. **P ≤ 0.01, ***P ≤ 0.001 ****P ≤ 0.0001. e Quantification of the types of mutagenic events (see “Methods” section for details) at breaks with 3′ ssDNA overhangs for the indicated mES cell-lines transfected with Cas9-N863A and sgRNAs targeting eGFP. GFP-negative (mutant) cells were sorted and used for targeted sequencing around the break-site, data represent the average of two independent clones per genotype. f Quantification of the extent of microhomology used for deletions and tandem duplications (TD) in the indicated genotypes. g Mutational signatures from (e) plotted as bars, relative to the Cas9-N863A-induced nicks (dashed lines), sorted by start position. The degree of blue coloring reflects the extent of microhomology. For TDs the duplicated sequence is plotted in orange. The height of each bar reflects the contribution of each outcome in the total amount of reads (Y-axis). DSB, double-strand break; MF, mutation frequency; delins, deletion insertion; snv, single nucleotide variant; TD, tandem duplication; TD+, tandem duplication with additional mutation (see “Methods” section); bp, base pair.