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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]

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]

In their Full Paper, R. K. A. van Schendel, W. Yang et al. combine the design of experiment approach with statistical analysis to provide a range of descriptive bias-free kinetic models for a representative ketone hydrogenation system. Keywords: Catalytic hydrogenation; Design of experiments; High-throughput experimentation; Reaction kinetics; Statistical analysis EN Catalytic hydrogenation Design of experiments High-throughput experimentation Reaction kinetics Statistical analysis 4857 4857 1 12/09/21 20211207 NES 211207 B The Front Cover b shows a "smart" statistic program that helps a researcher to navigate through complexity of practical catalytic systems and project a route to reliable and rapid assessment of kinetic parameters with resource-efficient experimentations. [Extracted from the article]

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., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA. In this study, we present a detailed and comprehensive genetic analysis of Cas9-induced DSB repair and T-DNA capture in the model plant Arabidopsis thaliana . We found that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) are both, and in part redundantly, acting on CRISPR-induced DSBs to produce very different mutational outcomes. We used newly developed CISGUIDE technology to establish that 8% of mutant alleles have captured T-DNA at the induced break site. In addition, we find T-DNA shards within genomic DSB repair sites indicative of frequent temporary interactions during TMEJ. Analysis of thousands of plant genome-T-DNA junctions, followed up by genetic dissection, further reveals that TMEJ is responsible for attaching the 3' end of T-DNA to a CRISPR-induced DSB, while the 5' end can be attached via TMEJ as well as cNHEJ. By identifying the mechanisms that act to connect recombinogenic ends of DNA molecules at chromosomal breaks, and quantifying their contributions, our study supports the development of tailor-made strategies toward predictable engineering of crop plants., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

The imine bond is among the most applied motifs in dynamic covalent chemistry. Although its uses are varied and often involve coordination to a transition metal for stability, mechanistic studies on imine exchange reactions so far have not included metal coordination. Herein, we investigated the condensation and transimination reactions of an Fe 2+ -coordinated diimine pyridine pincer, employing wB97XD/6-311G(2d,2p) DFT calculations in acetonitrile. We first experimentally confirmed that Fe 2+ is strongly coordinated by these pincers, and is thus a justified model ion. When considering a four-membered ring-shaped transition state for proton transfers, the required activation energies for condensation and transimination reaction exceeded the values expected for reactions known to be spontaneous at room temperature. The nature of the incoming and exiting amines and the substituents on the para-position of the pincer had no effect on this. Replacing Fe 2+ with Zn 2+ or removing it altogether did not reduce it either. However, the addition of two ethylamine molecules lowered the energy barriers to be compatible with experiment (19.4 and 23.2 kcal/mol for condensation and transimination, respectively). Lastly, the energy barrier of condensation of a non-coordinated pincer was significantly higher than found for Fe 2+ -coordinating pincers, underlining the catalyzing effect of metal coordination on imine exchange reactions., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)