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

Abstract

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 (Pol theta)-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 Arabidopsis thaliana requires Pol theta, in contrast to eMMEJ in animals. By reintroducing mutated versions of Pol theta into Pol theta-deficient plants we discovered that only the helicase activity of Pol theta is needed for eMMEJ; we demonstrate that plants lacking Pol theta'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 Pol theta 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