Gaetan Burgio

Background: The RNA bacteriophage MS2 is an RNA phage that infects the bacterium E. coli and is one of the most studied and prototypical model phages in molecular biology and microbiology. Previous research revealed complex translational control and fine-tuning for MS2 replication. However, the dynamics of its transcriptional activity and replication during the life cycles within the bacteria remain elusive. Methods: Here, we employed Nanopore Direct RNA sequencing (DRS) to investigate the transcriptome and epitranscriptome landscape of the MS2 in infected E. coli throughout multiple life cycles. Results: We discovered that MS2 phages sustain a high level of transcriptional activity required for replication. We found large amounts of subgenomic small transcripts from RNA degradation, Nanopore DRS bias, and transcripts containing the coat -encoding region, required for virion assembly. We found the error-prone activity of the MS2 replicase produced hybrid reads from the template-switching mechanism. We finally evidenced that RNA modification is conserved throughout the entire life cycle in full-length transcripts without the acquisition of new modifications, whereas small transcripts did acquire newly modified sites. The conserved sequence and secondary structure (U-rich hairpin) of Ψ installation sites were the most amenable to RNA modification, from potentially the host RluA-mediated installation. Conclusions: Overall, our investigation revealed a more complex transcriptional dynamics of MS2 phages than anticipated within E. coli to maintain its growth and replication under host pressure.

Cystinuria is a rare inherited disease characterized by increased urinary cystine levels resulting in the formation of cystine stones in the urinary tract. Mutations in the genes encoding the cystine transporter complex, SLC3A1 and SLC7A9, are the primary drivers of the disease. Current mouse models used to study cystinuria rely on gene deficiency or spontaneous mutations in mice that do not accurately reflect the pathogenic mutations found in humans. To overcome this limitation, we generated novel Slc7a9 ^G105R mice carrying the most common pathogenic single-point mutation in the SLC7A9 gene. Both male and female Slc7a9 ^G105R mice developed a cystinuria phenotype by 9 weeks of age, characterized by substantial cystine stone formation and increased urinary cystine, lysine, arginine, and ornithine. Slc7a9 ^G105R mice displayed distinct serum and urinary metabolite profiles mapped to dibasic amino acid pathways. Fecal metagenomics revealed that Slc7a9 ^G105R mice had a heterogeneous microbiota with altered functional pathways, including increased L-cysteine biosynthesis. Depletion of the microbiota with antibiotics did not impact cystine stone burden but reduced urinary tract inflammation. Prophylactic or therapeutic dietary supplementation with alpha-lipoic acid reduced stone burden and inflammation, but it also caused damage to the urothelium. Untargeted metabolomics analysis following alpha-lipoic acid supplementation identified metabolites that can increase cystine solubility, reduce inflammation, and damage epithelial cells. Correlation analysis revealed novel serum biomarkers of stone burden, including blood urea nitrogen, 2-hydroxybutyric acid, 2-amino-2-thiazoline-4-carboxylic acid, and indole-3-acetylglycine. Collectively, the Slc7a9 ^G105R mutant mouse model offers a precise, rapid-onset, and translational platform for investigating cystinuria pathogenesis and evaluating potential therapeutic strategies. Translational statement: The development of a novel knock-in mouse model carrying the most common pathogenic point mutation in the human SLC7A9 gene provides a clinically relevant and translationally valuable platform for investigating cystinuria pathogenesis and testing emerging therapies. This model represents the closest possible approximation of human SLC7A9 -mediated cystinuria, enabling rigorous preclinical evaluation of small molecules and gene therapies. It has also facilitated the identification of candidate biomarkers for cystine stone burden and treatment response, which are urgently needed to improve disease monitoring and clinical decision-making. The next critical step is to validate these biomarkers in human cystinuria cohorts to support their clinical translation.

The trans cleavage activity of Cas12a has been extensively used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions in the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficacy. Overall, this study provides a blueprint for rationally engineering the DNase activities of Cas12a.

Nanopore direct RNA sequencing promises to decode the epitranscriptome by detecting multiple modifications on individual RNA molecules, but its potential for biological discovery is hampered by high false-positive rates. We present SWARM, an AI-based framework designed to overcome this fundamental limitation. Its key innovation is a crosstalk-aware training strategy that incorporates non-target modifications and orthogonally validated cellular signals, enabling high-precision detection of m6A, pseudouridine (Ψ), and m5C at single-nucleotide and single-molecule resolution. Using rigorous in vitro and cellular RNA benchmarks, SWARM outperforms existing tools and maintains strong agreement with orthogonal methods. Applying SWARM across mammalian tissues reveals thousands of novel modification sites with confirmed motifs and localisation patterns. Our high-resolution multi-tissue modification map revealed no evidence of widespread m6A-Ψ interplay in predominant writer contexts, challenging models of a coordinated epitranscriptomic code. We further discovered a previously unrecognised splicing-shaped mode of Ψ deposition, whereby TRUB1-mediated pseudouridylation preferentially occurs after exon-exon ligation, consistent with local RNA structure stabilisation. SWARM provides a robust, universally applicable tool for epitranscriptome discovery.

The trans cleavage activity of type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas12a system has been widely used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers, and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions of the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficiency. Overall this study provides a blueprint for future rational engineering of Cas12a nucleases for their trans cleavage activities. Graphical

CRISPR-Cas12a mediates RNA-guided cleavage of double-stranded DNA in cis, after which it remains catalytically active and non-specifically cleaves single-stranded DNA in trans. Native host defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well-characterized Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in Escherichia coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-Nuc interactions. We generated and tested REC2 and Nuc mutants, including a hitherto unstudied ‘Nuc-loop’, integrity of which is critical for the function of Cas12. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.

The RNA bacteriophage MS2 is an RNA phage that infects the bacterium E. coli and is one of the most studied and prototypical model phages in molecular biology and microbiology. Previous research revealed complex translational control and fine-tuning for MS2 replication. However, the dynamics of its transcriptional activity and replication during the life cycles within the bacteria remain elusive. Here, we employed Nanopore Direct RNA sequencing (DRS) to investigate the transcriptome and epitranscriptome landscape of the MS2 in infected E. coli throughout multiple life cycles. We discovered that MS2 phages sustain a high level of transcriptional activity required for replication. We found large amounts of subgenomic small transcripts from RNA degradation, Nanopore DRS bias, and transcripts containing the coat -encoding region, required for virion assembly. We found hybrid reads due to the error-prone activity of the MS2 replicase complex by the template-switching mechanism. We evidenced that RNA modification is conserved throughout the entire life cycle in full-length transcripts without the acquisition of new modifications, whereas small transcripts did acquire newly modified sites. The conserved sequence and secondary structure (U-rich hairpin) of Ψ installation sites were the most amenable to RNA modification, and one site aligned well with host RluA-mediated installation. Overall, our investigation reveals a more complex transcriptional dynamics of MS2 phages than anticipated within E. coli to maintain its growth and replicate under host pressure.

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis , after which it remains catalytically active and non-specifically cleaves ssDNA in trans . Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli , and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied ‘NUC loop’, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical

Context: The obesity epidemic parallels an increasing type 1 diabetes incidence, such that westernized diets, containing high fat, sugar, and/or protein, through inducing nutrient-induced islet β-cell stress, have been proposed as contributing factors. The broad-spectrum neutral amino acid transporter (B0AT1), encoded by Slc6a19, is the major neutral amino acids transporter in intestine and kidney. B0AT1 deficiency in C567Bl/6J mice causes aminoaciduria, lowers insulinemia, and improves glucose tolerance. Objective: We investigated the effects of standard rodent chow (chow), high-fat high-sucrose (HFHS), and high-fat high-protein (HFHP) diets, in addition to B0AT1 deficiency, on the diabetes incidence of male nonobese diabetic (NOD/ShiLtJArc (NOD)) mice. Methods: Male NOD.Slc6a19+/+ and NOD.Slc6a19−/− mice were fed chow, HFHS and HFHP diets from 6 to 24 weeks of age. A separate cohort of male NOD mice were fed the three diets from 6-30 weeks of age. Body weight and fed-state blood glucose and plasma insulin were monitored, and urinary amino-acid profiles, intraperitoneal glucose tolerance, diabetes incidence, pancreatic islet number, insulitis scores and beta-cell mass were measured. Results: The incidence of diabetes and severe glucose intolerance was 3.8% in HFHS-fed, 25.0% in HFHP-fed, and 14.7% in chow-fed mice, with higher pancreatic islet number and lower insulitis scores in HFHS-fed mice. B0AT1 deficiency had no effect on diabetes incidence, but curtailed HFHS-induced excessive weight gain, adipose tissue expansion, and hyperinsulinemia. In HFHP-fed mice, B0AT1 deficiency significantly increased pancreatic β-cell clusters and small islets. Male NOD mice that did not develop autoimmune diabetes were resistant to diet-induced hyperglycemia. Conclusion: Dietary composition does, but B0AT1 deficiency does not, affect autoimmune diabetes incidence in male NOD mice. B0AT1 deficiency, however, reduces diet-induced metabolic dysfunction and in HFHP-fed mice increases pancreatic β-cell clusters and small islets.