The length and sequence of the primer binding site (PBS) are critical for efficient prime editing, and its intramolecular complementarity with the prime editing guide RNA (pegRNA) spacer is a major drawback. We investigated the effects of these factors by literature analyses and by testing over 300 modified pegRNAs with weakened PBS-spacer interactions. It has been suggested that the effective PBS length for plasmid-delivered pegRNAs without end protection is considerably longer than what efficient priming requires due to exonuclease digestion of the PBS ends; however, analysing literature data of over 3000 pegRNAs revealed no significant shift in the optimal PBS length for epegRNAs compared to conventional pegRNAs. We also found improvement in editing efficiency with up to seven-fold when mismatches were introduced in the spacer or PBS sequence disrupting complementarity, although this effect is more pronounced with non-optimal PBS lengths. A combination of spacer mismatches and PBS deletions led to further editing improvements, even compared to the optimal PBS, although finding the best combination requires extensive optimization. Here, we achieved near-optimal editing efficiency in the majority of cases without the need for prior pegRNA optimization by using SPELL (Streamlined Prime Editing with fixed-Length PBS Leverage), a prime editing approach that employs a 17–20 nucleotide-long PBS with a single nucleotide deletion.

Forming effective responses to threatening stimuli requires the adequate and coordinated emergence of stress-related internal states. Such ability depends on early-life experiences and, in connection, the adequate formation of neuromodulatory systems, particularly serotonergic signaling. Here, we assess the serotonergic background of experience-dependent behavioral responsiveness employing a zebrafish ( Danio rerio ) model. For the first time, we have characterized a period during the behavioral metamorphosis in which zebrafish are highly reactive to their environment. Absence of social stimuli during this phase established by isolated rearing fundamentally altered the behavioral phenotype of post-metamorphic zebrafish in a challenge-specific manner, partially due to a decline in responsiveness and an inability to develop stress-associated arousal state. In line with this, isolation differently affected whole-brain 5-HT signaling in resting and stress-induced conditions, an effect that was present at the level of the dorsal pallium and was negatively associated with responsiveness. Administration of the 5HT1AR partial agonist buspirone prevented the isolation-induced serotonin response to novelty in the forebrain and rescued stress-induced arousal along with challenge-induced behaviors, which altogether indicates a functional connection between these changes. In summary, there is a consistent negative association between behavioral responsiveness and serotonergic signaling in zebrafish, which is well recognizable through the modifying effects of developmental perturbation and pharmacological manipulations as well. Our results imply a conserved serotonergic mechanism that context-dependently modulates environmental reactivity and is highly sensitive to experiences acquired during a specific early-life time-window, a phenomenon that was previously only suggested in mammals. Significance statement: The ability to respond to challenges is a fundamental factor in survival. We show that zebrafish that lack appropriate social stimuli in a sensitive developmental period show exacerbated alertness in non-stressful conditions while failing to react adequately to stressors. This shift is reflected inversely by central serotonergic signaling, a system that is implicated in numerous mental disorders in humans. Serotonergic changes in brain regions modulating responsivity and behavioral impairment were both prevented by the pharmacological blockade of serotonergic function. These results imply a serotonergic mechanism in zebrafish that transmits early-life experiences to the later phenotype by shaping stress-dependent behavioral reactivity, a phenomenon that was previously only suggested in mammals. Zebrafish provide new insights into early-life-dependent neuromodulation of behavioral stress-responses.

RecQ helicases - also known as the ‘guardians of the genome’ - play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish ( Danio rerio ) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51 , however, blm appears to effect its function independent of tp53 . Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

The use of nanoscale materials has expanded rapidly in recent decades, with titanium dioxide (TiO₂) nanoparticles among the most widely produced. Their increasing environmental release raises concerns about ecosystem-level effects. A key mechanism of toxicity is the generation of reactive oxygen species (ROS), although these effects strongly depend on particle properties, including crystalline form, size, morphology, surface characteristics, and exposure conditions. Here, we assessed and compared the ecological impacts of anatase and rutile TiO₂ nanoparticles across prokaryotic and eukaryotic systems, including aquatic microbial communities, microbial cultures, plant bioassays (Sinapis alba, Triticum aestivum), the SOS Chromotest, and zebrafish (Danio rerio) assays. Nano-TiO₂ exposure markedly restructured freshwater microbial communities by suppressing sensitive taxa (e.g., Actinomycetota, Flavobacterium, and Limnohabitans) while enriching more tolerant genera such as Pseudomonas, Sediminibacterium, Haliscomenobacter, and Hydrogenophaga. These shifts likely reflect differences in cell-envelope structure, biofilm formation, and antioxidant capacity. The two investigated TiO₂ nanoparticle types showed distinct biological effects: rutile was associated with more pronounced microbial community shifts and bacterial cell damage, whereas anatase caused stronger responses in plant assays, particularly by impairing hypocotyl growth and plant water balance. Besides the limited acute genotoxicity revealed by the SOS Chromotest, TiO₂ nanoparticles did not significantly affect survival or final larval body length in the zebrafish embryo assay under the tested conditions; however, the hatching delay observed at the highest concentration indicated a sublethal developmental effect. Overall, our results show that TiO₂ nanoparticle toxicity cannot be generalized across biological systems and suggest that biological responses depend on the combined influence of particle characteristics and organism-specific structural and physiological traits.