Fig. 1: Cleavage of the IGS region of the 45S rDNA repeats in Arabidopsis using SaCas9.

a The 45S rDNA in Arabidopsis is arranged in up to 750 repeats, in a head-to-tail-fashion. Each repeat consists of a unit of three rRNA genes (18S, 5.8S, and 25S), separated by a variable intergenic spacer (IGS) region. The individual rRNA genes are separated by two highly conserved internal transcribed spacers (ITS). The triangles represent the cleavage sites for SaCas9 within the repeats. By targeting the variable IGS region (petrol), only a subset of repeats was cleaved. b The break site was analyzed by deep sequencing. Only a very small fraction of mutations was identified as InDels (yellow) or insertions (blue), whereas over 95% were deletions (green). Further analysis of the reads, including the deletions, showed that 94% of these were small deletions with a size of 10 bp or smaller. The deletion pattern of these small deletions revealed that no microhomologies were used for the repair (red). c Detailed illustration of exemplary junctions with deletions in Col-0 plants on the sequence level. d Cleavage of the IGS region in DNA repair mutants revealed that only the cNHEJ mutants, ku70-1 and lig4-5, but not the HR mutant, rad54-1, responded highly sensitive, resulting in a drastically reduced survival rate (n = 3 biologically independent experiments). Data are presented as normalized mean values ± SD. Statistical differences were calculated using the two-tailed t-test with unequal variances: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source data file.