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The fidelity of genetic information is essential for cellular function and viability. DNA double-strand breaks (DSBs) pose a significant threat to genome integrity, necessitating efficient repair mechanisms. While the predominant repair strategies are usually accurate, paradoxically, error-prone pathways also exist. This review explores recent advances and our understanding of microhomology-mediated end joining (MMEJ), an intrinsically mutagenic DSB repair pathway conserved across organisms. Central to MMEJ is the activity of DNA polymerase theta (Polθ), a specialized polymerase that fuels MMEJ mutagenicity. We examine the molecular intricacies underlying MMEJ activity and discuss its function during mitosis, where the activity of Polθ emerges as a last-ditch effort to resolve persistent DSBs, especially when homologous recombination is compromised. We explore the promising therapeutic applications of targeting Polθ in cancer treatment and genome editing. Lastly, we discuss the evolutionary consequences of MMEJ, highlighting its delicate balance between protecting genome integrity and driving genomic diversity. [ABSTRACT FROM AUTHOR]

SUMMARY: With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double‐strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB‐end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9‐induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non‐homologous end‐joining (NHEJ) produces 70% of mutations, whereas polymerase theta‐mediated end‐joining (TMEJ) generates 30%, indicating that DSB‐end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T‐DNA molecules as does Cas9. Long‐read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non‐symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill‐in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences. [ABSTRACT FROM AUTHOR]

SUMMARY: Precise genetic modification can be achieved via a sequence homology‐mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta‐mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology‐directed process. Here, by monitoring homology‐driven gene conversion in plants with CRISPR reagent and donor sequences pre‐integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end‐joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ‐deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T‐DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants. [ABSTRACT FROM AUTHOR]

Small tandem DNA duplications in the range of 15 to 300 base‐pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9‐based experimental data from different model systems. Recent work points to fill‐in synthesis at double‐stranded DNA breaks with complementary sequences, regulated by end‐joining mechanisms, to account for small tandem duplications. We will review the prevalence of small tandem duplications in the population, and we will speculate on the potential sources of DNA damage that could give rise to this mutational signature. With the development of novel algorithms to analyse sequencing data, small tandem duplications are now more frequently detected in the human genome and identified as oncogenic gain‐of‐function mutations. Understanding their origin could lead to optimized treatment regimens to prevent therapy‐induced activation of oncogenes and might expose novel vulnerabilities in cancer. [ABSTRACT FROM AUTHOR]

Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations, msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lac[Z.sup.+] patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.

SUMMARY: Agrobacterium tumefaciens‐mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T‐DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)‐mediated end joining (TMEJ). Targeting of the T‐DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T‐DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ‐proficient plants we predominantly found GT events accompanied by random T‐DNA integrations, GT events obtained in the teb mutant background lacked additional T‐DNA copies, corroborating the essential role of Polθ in T‐DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high‐quality GT events in crop plants. Significance Statement: We find that homologous recombination‐mediated gene targeting (GT) in polymerase theta (Polθ)‐deficient Arabidopsis exclusively produces plants that contain true GT events and are devoid of random T‐DNA integration. These observations corroborate the crucial role for Polθ in T‐DNA integration, and reveal that Polθ‐mediated end joining is essential for ectopic GT, which is a prominent outcome in Polθ‐proficient plants. [ABSTRACT FROM AUTHOR]