Biology subjects

Chalmers, R.

Publications and source records attributed to Chalmers, R..

7 recordsLinked to original sources

bioRxiv2026

Cas1 epistasis tunes conformational coupling to enhance CRISPR adaptation

CRISPR adaptation requires Cas1-Cas2 to capture prespacers, undergo conformational rearrangement and catalyse integration, but how these steps are coupled remains unclear. Using nine hyperactive Escherichia coli Cas1 substitutions as perturbational probes, we identified a prespacer-coupling module intersecting an interior conformational-coupling module. Single substitutions increased adaptation up to sixfold, whereas combinatorial reassortment generated a genotype with 103-fold greater activity than wild type. Genotype-network analysis and quantitative reconstruction revealed strong background-dependent epistasis: the same substitution could enhance activity in one genotype but impair it in another, and high activity emerged only from compatible combinations spanning both modules. These findings indicate that Cas1-Cas2 activity is constrained by compatibility among changes distributed across the Cas1 dimer, rather than by optimization of individual catalytic or DNA-binding interactions. We propose that coordinated tuning of prespacer engagement and conformational coupling governs Cas1-Cas2 activity during CRISPR adaptation.

biochemistry↗

bioRxiv2026

RecA status determines SOS- and RecBCD-dependent outcomes in CRISPR-Cas adaptation

CRISPR-Cas immunity depends on integrating DNA fragments from invading elements, yet how this process is tuned by host physiology remains poorly understood. Here, we investigate the relationship between CRISPR adaptation and the bacterial SOS DNA damage response in Escherichia coli using a highly sensitive colony-based adaptation assay. Blocking SOS with a lexA3 allele suppresses adaptation, while paradoxically, deleting recA enhances it. These findings indicate that RecA contributes positively to adaptation indirectly, through LexA cleavage and induction of SOS-regulated functions, while complete loss of RecA is associated with a RecBCD-dependent DNA-processing state that favours successful adaptation. The RecA inhibitors RecX and PsiB did not phenocopy {triangleup}recA, indicating that inhibitor-mediated perturbation of RecA in RecA-proficient cells and complete loss of RecA have different consequences for adaptation. Genome-wide maps of recovered spacers show that RecA and LexA reshape the relative distribution of chromosomal and plasmid-derived spacers, linking CRISPR adaptation to replication and DNA repair dynamics. Although Cas1-Cas2 catalyses spacer integration autonomously in vitro, our findings suggest that successful adaptation in vivo emerges from interactions between spacer acquisition, DNA repair, and bacterial stress physiology, embedding CRISPR adaptation within cellular networks that balance immune protection with the risk of autoimmunity.

microbiology↗

bioRxiv2025

Parapipe: a Pipeline for Handling Parasite NGS Datasets and its Application to Cryptosporidium

0.0Cryptosporidium, a protozoan parasite of significant public health concern, is responsible for severe diarrheal diseases, particularly in immunocompromised individuals and young children in resource-limited settings. Analysis of whole genome next generation sequencing (NGS) data is a critical next step in improving our understanding of Cryptosporidium epidemiology, transmission dynamics, and genetic diversity. However, effective analysis of NGS data in a public health context necessitates the development of robust, validated bioinformatics tools. Here, we present Parapipe, a modular ISO accreditable bioinformatics pipeline designed for high-throughput processing and analysis of Cryptosporidium NGS datasets. Built using Nextflow DSL2 and containerized with Singularity, Parapipe is portable, scalable, and capable of end-to-end analyses, including quality control, variant calling, multiplicity of infection (MOI) investigations and phylogenomic clustering analysis. Using both simulated and real-world datasets, we demonstrate Parapipes ability to resolve genetic heterogeneity, identify mixed infections, and generate high-resolution phylogenomic insights. Here, we use it to carry out a comparison between whole genome single nucleotide polymorphism (wgSNP) typing and the conventionally used gp60 molecular typing scheme. Compared to existing pipelines, Parapipe uniquely integrates MOI analysis, enabling the differentiation of mixed infections and supporting epidemiological investigations. Parapipes design facilitates integration with geographic, demographic, epidemiological and environmental data, enhancing its utility for tracking transmission pathways and outbreak sources. Parapipe represents a significant advance in utilising genomics for public health surveillance of Cryptosporidium, offering a streamlined and reproducible framework for analysis with potential application to other pathogenic protozoa. By automating complex workflows and enabling detailed genomic characterization, Parapipe provides a valuable tool for public health agencies and researchers, supporting efforts to mitigate the global burden of cryptosporidiosis.

bioinformatics↗

bioRxiv2023

Comparative genomics reveals the emergence of an outbreak-associated Cryptosporidium parvum population in Europe and its spread to the USA

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g. IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water-and food-borne outbreaks. In this study, we generated new whole genome sequence (WGS) data from 123 human-and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China and the USA (n=72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analysed a dataset from 141 isolates from the zoonotic groups IIa (n=119) and IId (n=22). Based on 28,047 high quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: isolates from China (IId) and Egypt (IIa and IId) formed population 1, a minority of European isolates (IIa and IId) formed population 2, while the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the USA (IIa) clustered in population 3. Based on analyses of the population structure, population genetics and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the UK, reach the USA where it also expanded. In addition, genetic exchanges between different populations led to the formation of mosaic genomes. The reason(s) for the successful spread of population 3 remained elusive, although genes under selective pressure uniquely in this population were identified.

genomics↗

bioRxiv2019

In vitro transposon targeting using a catalytically inactive Cas9

Homology directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive candidate for targeting a transposase fusion-protein because of its high specificity and affinity for its binding site. Here we demonstrate efficient Cas9 targeting of a mariner transposon. Targeted integrations were tightly constrained at two adjacent TA dinucleotides about 20 bp to one side of the gRNA binding site. Biochemical analysis of the nucleoprotein complexes demonstrated that the transposase and Cas9 moieties of the fusion protein can bind their respective substrates independently. In the presence of the Cas9 target DNA, kinetic analysis revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase behaved normally and was proficient for integration in vitro and in vivo.

biochemistry↗

bioRxiv2018

SETMAR functions in illegitimate DNA recombination and non-homologous end joining

In anthropoid primates, SETMAR is a fusion between a methyltransferase gene and a domesticated DNA transposase. SETMAR has been found to be involved in several cellular functions including regulation of gene expression, DNA integration and DNA repair. These functions are thought to be mediated through the histone methyltransferase, the DNA binding and the nuclease activities of SETMAR. To better understand the cellular roles of SETMAR, we generated several U2OS cell lines expressing either wild type SETMAR or a truncated or mutated variant. We tested these cell lines with in vivo plasmid-based assays to determine the relevance of the different domains and activities of SETMAR in DNA integration and repair. We found that expressing the SET and MAR domains, but not wild type SETMAR, partially affect DNA integration and repair. The methyltransferase activity of SETMAR is also needed for an efficient DNA repair whereas we did not observe any requirement for the putative nuclease activity of SETMAR. Overall, our data support a non-essential function for SETMAR in DNA integration and repair.

molecular biology↗

bioRxiv2018

Development of a papillation assay using constitutive promoters to find hyperactive transposases

BackgroundTransposable elements (TEs) form a diverse group of DNA sequences encoding functions for their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, their use is sometimes limited because their activity is auto-regulated to allow them to cohabit within their hosts without causing excessive genomic damage. To overcome these limitations, it is important to develop efficient and simple screening assays for hyperactive transposases.\n\nResultsTo widen the range of transposase expression normally accessible with inducible promoters, we have constructed a set of vectors based on constitutive promoters of different strengths. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. We observed the highest rate of transposition with the weakest promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase dimer interface and of covalently linking two transposase monomers in a single-chain dimer. We also tested the severity of mutations in the lineage leading to the human SETMAR gene, in which one copy of the Hsmar1 transposase has contributed a domain.\n\nConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. We also found that mutations in the Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

molecular biology↗