SRP309183 PRJNA706449 Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks DNA double strand breaks (DSBs) broadly represent the most deleterious and mutagenic lesion facing the genome, and their accurate repair is essential for proper genome stability and cellular integrity. DSB repair generally proceeds through either homologous recombination or classical nonhomologous end joining, A third repair mechanism, called alternative end joining (alt-EJ), also repairs DSBs and its signatures are associated with homologous recombination-deficient cancer genomes. We have previously proposed a model for alt-EJ whereby short DNA repeats near a DSB (primer repeats) can anneal to form secondary structure intermediates that prime limited de novo synthesis. The nascent DNA then anneals with microhomologous sequences on the other side of the break. This model, termed synthesis dependent microhomology mediated end joining (SD-MMEJ), explains many of the alt-EJ repair products recovered following break formation by nucleases. However, the sequence-specific factors that influence the progression of SD-MMEJ repair outcomes remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through characterization of repair products at Cas9-induced DSBs in more than 1100 different sequence contexts. Through computational analysis, we find evidence at single nucleotide resolution for several factors that drive successful SD-MMEJ repair. We characterize primer repeat and microhomology usage according to length, GC content, and proximity to the break. Interestingly, the flexibility of DNA between primer repeats is an important determinant of SD-MMEJ success. Finally, we find that templates for existing microhomologies found near the break site drive successful SD-MMEJ. The methods described herein represent a streamlined computational pipeline that can be utilized to analyze preferred mechanisms of alt-EJ repair in any sequence context.