Duplication of existing sequences is a major mechanism of genome evolution. It has been previously shown that duplications can occur by replication slippage, unequal sister chromatid exchange, homologous recombination, and aberrant double-strand break-induced synthesis-dependent strand annealing reactions. In a recent study, the abundant presence of short direct repeats was documented by comparative bioinformatics analysis of different rice genomes, and the hypothesis was put forward that such duplications might arise due to the concerted repair of adjacent single-strand breaks (SSBs). Applying the CRISPR/Cas9 technology, we were able to test this hypothesis experimentally in the model plant Arabidopsis thaliana. Using a Cas9 nickase to induce adjacent genomic SSBs in different regions of the genome (genic, intergenic, and heterochromatic) and at different distances (∼20, 50, and 100 bps), we analyzed the repair outcomes by deep sequencing. In addition to deletions, we regularly detected the formation of direct repeats close to the break sites, independent of the genomic context. The formation of these duplications as well as deletions may be associated with the presence of microhomologies. Most interestingly, we found that even the induction of two SSBs on the same DNA strand can cause genome alterations, albeit at a much lower level. Because such a scenario reflects a natural step during nucleotide excision repair, and given that the germline is set aside only late during development in plants, the repair of adjacent SSBs indeed seems to have an important influence on the shaping of plant genomes during evolution. T he opportunity to experimentally investigate the outcome of double-strand break (DSB) repair in eukaryotic organisms was enabled by the use of site-specific endonucleases targeting unique genomic sequences. Early work using the meganuclease I-SceI more than 20 y ago demonstrated that the induction of a DSB leads to enhanced homologous recombination (HR) in multicellular eukaryotes (1). This finding led to the application of I-SceI as a tool for detailed analysis of genomic changes that might occur due to DSB repair via nonhomologous end-joining (NHEJ) in plants. Thus, it was possible to demonstrate that along with inducing various kinds of deletions (2, 3), DSB repair also can be associated with the integration of T-DNA, as well as with the copying of genomic sequences into the break (4, 5).Recently, a novel type of programmable nuclease was introduced by the discovery of the molecular mechanism of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) system (6, 7). In this system, the endonuclease Cas9 is guided by two specialized RNAs (CRISPR RNA and transactivating CRISPR RNA) via direct base-pairing to bind and cleave invading harmful DNA. It has been demonstrated in vitro and in vivo that by fusing the two RNAs to a so-called singleguide RNA (sgRNA), the system can serve as a highly efficient and precisely programmable nuclease for genome eng...