For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. We have recently demonstrated for two of the most widely used mutagens in C. elegans, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs) 1, 2. This discovery allowed us to survey many thousands of available deletion alleles, which reveals a step-wise and versatile model for the in vivo mechanism of TMEJ, explaining the molecular nature of mutagen-induced deletion alleles 3. We also found that CRISPR/Cas9-induced genomic changes are exclusively generated through POLQ action, refuting a previously assumed requirement for NHEJ in their formation; Unlike somatic cells, which use NHEJ to repair DNA breaks, germ cells use an alternative route 4. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification. New insights into the mechanism of genome engineering, genome maintenance and evolution will be discussed. 1. Koole,W. et al. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 5, 3216 (2014). 2. Roerink,S.F., van Schendel,R., & Tijsterman,M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954-962 (2014). 3. van Schendel R., van Heteren J., Welten,R., & Tijsterman,M. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining. PLoS. Genet. 12,
e1006368 (2016). 4. van Schendel,R., Roerink,S.F., Portegijs,V., van den Heuvel,S., & Tijsterman,M. Polymerase Theta is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat. Commun. 6, 7394 (2015).