Division of labor within polymerase theta in repair of CRISPR-induced DNA breaks in .

To develop efficient strategies for precise mutagenesis in plants, it is crucial to characterize the mechanisms involved in the repair of CRISPR-induced double strand breaks (DSBs). Polymerase theta (Polθ)-mediated end joining (TMEJ) and classical nonhomologous end joining are key pathways that generate a wide array of mutations during DSB repair. To direct repair towards more predictable outcomes, we examined the impact of direct repeats flanking DSBs, which may trigger extended microhomology-mediated end joining (eMMEJ). Unexpectedly, we found that eMMEJ in requires Polθ, in contrast to eMMEJ in animals. By reintroducing mutated versions of Polθ into Polθ-deficient plants we discovered that only the helicase activity of Polθ is needed for eMMEJ; we demonstrate that plants lacking Polθ's polymerase domain are incapable of TMEJ and are resistant to TMEJ-dependent T-DNA integration but still support extended microhomology-guided DSB repair at genomic sites with direct repeats. These findings reveal species-specific functionality of Polθ and point to functional divergence in TMEJ across species. Additionally, these insights provide new opportunities to direct targeted mutagenesis in plants toward single, predictable outcomes, paving the way for more efficient crop engineering. Classification: Biological, Health, and Medical Sciences.