Deficiency of both classical and alternative end‐joining pathways leads to a synergistic defect in double‐strand break repair but not to an increase in homology‐dependent gene targeting in Arabidopsis

Merker, Laura; Feller, Laura; Dorn, Annika; Puchta, Holger

doi:10.1111/tpj.16604

Cited by 3 publications

(1 citation statement)

References 89 publications

(130 reference statements)

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“…Alternative end-joining (alt-EJ) pathways operate in plants, including DNA polymerase theta (POLQ) mediated end-joining (TMEJ) [ 157 ], although functions in seeds are not well characterised. However, recently a ku70 polq double mutant was reported to have reduced germination [ 158 ].…”

Section: Dna Damage and Repairmentioning

confidence: 99%

Seed longevity and genome damage

Waterworth,

Balobaid,

West

2024

Bioscience Reports

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Seeds are the mode of propagation for most plant species and form the basis of both agriculture and ecosystems. Desiccation tolerant seeds, representative of most crop species, can survive maturation drying to become metabolically quiescent. The desiccated state prolongs embryo viability and provides protection from adverse environmental conditions, including seasonal periods of drought and freezing often encountered in temperate regions. However, the capacity of the seed to germinate declines over time and culminates in the loss of seed viability. The relationship between environmental conditions (temperature and humidity) and the rate of seed deterioration (ageing) is well defined, but less is known about the biochemical and genetic factors that determine seed longevity. This review will highlight recent advances in our knowledge that provide insight into the cellular stresses and protective mechanisms that promote seed survival, with a focus on the roles of DNA repair and response mechanisms. Collectively, these pathways function to maintain the germination potential of seeds. Understanding the molecular basis of seed longevity provides important new genetic targets for the production of crops with enhanced resilience to changing climates and knowledge important for the preservation of plant germplasm in seedbanks.

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Section: Dna Damage and Repairmentioning

confidence: 99%

Seed longevity and genome damage

Waterworth,

Balobaid,

West

2024

Bioscience Reports

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Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana

Kralemann,

van Tol,

Hooykaas

et al. 2024

The Plant Journal

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SUMMARYPrecise 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.

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Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana

Li,

Wei,

Cheng

et al. 2024

BMC Plant Biol

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Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

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Deficiency of both classical and alternative end‐joining pathways leads to a synergistic defect in double‐strand break repair but not to an increase in homology‐dependent gene targeting in Arabidopsis

Cited by 3 publications

References 89 publications

Seed longevity and genome damage

Seed longevity and genome damage

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana

Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana

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