bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.10.26.761776

Development of a CRISPR-Cas9 genome editing system for the extremely thermophilic bacterium Anaerocellum bescii

Galindo, J. L.·Conway, J. M.
View original source ↗Export metadata ↓

Abstract

Anaerocellum (f. Caldicellulosiruptor) bescii is an extremely thermophilic (Topt 75-78 {degrees}C) anaerobic bacterium and is also a highly efficient degrader of lignocellulose, making it a promising platform for metabolic engineering. To date, chromosomal genetic manipulations in A. bescii have required multistep procedures involving homologous recombination with integrating vectors and counterselection. Here, we demonstrate the first implementation of CRISPR-Cas9 genome editing in A. bescii facilitated by a thermostable Cas9 enzyme from Geobacillus stearothermophilus (GeoCas9). Delivery of heterologous CRISPR machinery via a replicating vector enabled us to individually and sequentially delete genes for lactate dehydrogenase (ldh) and pyrE in A. bescii. We then deployed this system to delete a novel target, tryptophan synthase (trpBA), generating a tryptophan auxotrophic strain of A. bescii. Using this strain, we show trpBA complementation enables selection and maintenance of a replicating vector expressing a heterologous enzymatic reporter gene.

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Galindo, J. L., Conway, J. M.. 2026-10-28. Development of a CRISPR-Cas9 genome editing system for the extremely thermophilic bacterium Anaerocellum bescii. https://doi.org/10.64898/2026.10.26.761776

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Tissue-specific transcriptomic responses to early Toxoplasma gondii infection in sheep during congenital toxoplasmosis

Toxoplasma gondii is a widespread zoonotic pathogen of major One Health concern, capable of causing congenital toxoplasmosis in both humans and sheep. Despite its impact, early host-parasite molecular interactions during acute infection in pregnant hosts remain largely unexplored. In this study, we characterized the transcriptomic responses of key tissues involved in early infection (the small intestine, mesenteric lymph nodes, and placenta) in pregnant ewes experimentally infected with T. gondii strain TgShSp1. RNA-seq analysis revealed pronounced, tissue-specific transcriptional changes at 6 days post-infection (dpi), particularly in the mesenteric lymph nodes, characterized by activation of interferon- and cytokine-mediated immune pathways. In contrast, transcriptional responses at 3 dpi were limited, consistent with a silent phase of parasite dissemination. Despite the absence of detectable parasite DNA, placentomes showed modest interferon-associated responses and upregulation of DNA repair and cellular stress pathways. These changes coincided with marked suppression of cell-cycle progression and extracellular matrix remodeling alongside inhibition of angiogenesis and hypoxia pathways. Altogether, early disruption of placental vascular function and tissue remodeling may contribute to fetal loss. In summary, these findings provide new insights into early host responses to T. gondii infection in a pregnant host and identify molecular mechanisms that may predispose to early fetal death during ovine congenital toxoplasmosis.

microbiology↗
bioRxiv2026

Chikungunya virus live-cell reporter systems reveal cell-type-dependent antiviral responses to ribavirin

Chikungunya virus (CHIKV) remains a significant global public health threat, yet antiviral discovery is limited by experimental systems that provide little mechanistic resolution and rely heavily on transformed cell lines of uncertain physiological relevance. Here, we developed complementary live-cell CHIKV reporter systems comprising a dual-fluorescent subgenomic replicon and fluorescent infectious viruses that enable real-time monitoring of distinct stages of the viral replication cycle. By coupling an nsP3-tagged reporter to genomic RNA translation and replication with a subgenomic reporter of structural gene expression, we achieved temporal resolution of early and late replication events in living cells. Functional validation using replication-defective and subgenomic RNA-deficient mutants confirmed that the system distinguishes between viral RNA replication and downstream structural gene expression. Application of these reporters revealed distinct antiviral responses across transformed and physiologically relevant human cell models. Ribavirin and the nsP2 inhibitor RA-0002034 potently inhibited CHIKV replication in transformed cell lines. Unexpectedly, ribavirin activity was markedly diminished in primary human skeletal muscle myoblasts and dermal fibroblasts, despite robust antiviral effects in commonly used screening cell lines. Mechanistic studies showed that ribavirin-mediated inhibition was rescued by guanosine but not inosine supplementation and was recapitulated by mycophenolic acid, consistent with guanine nucleotide depletion as the predominant antiviral mechanism under the conditions tested. Notably, inhibition through this pathway was substantially less effective in primary human cells. Together, these findings establish versatile live-cell CHIKV reporter systems for mechanistic studies of viral replication and reveal striking cell-type-dependent differences in response to guanine nucleotide depletion. Our results demonstrate that host-cell context can profoundly influence both CHIKV replication and antiviral susceptibility, highlighting the importance of physiologically relevant models for understanding virus-host interactions and antiviral mechanism.

microbiology↗
bioRxiv2026

A droplet-to-data workflow for antimicrobial screening of complex matrices

Screening plant extracts and other complex natural matrices for antibacterial activity is complicated by optical interference and population averaging in conventional bulk assays. Droplet-based assays mitigate interference while enabling high-throughput assessment of bacterial subpopulations. However, analyzing the resulting image data involves difficult image processing, classification, and interpretation. Here, we present a democratized workflow for antimicrobial screening in complex matrices that combines a polydisperse droplet-based assay with EasyFlow 2.0 (EF2), our updated droplet image analysis platform. EF2 offers stable containerized deployment, expanded polydisperse droplet support, automated thresholding and MIC estimation, LLM-assisted data exploration, and import of other software outputs such as CellProfiler (CP) measurements and CellProfiler Analyst (CPA) classification models. As proof of concept, we applied this workflow to assess the antibacterial and aggregation-modulating effects of chemically distinct extracts of Taraxacum officinale, Rhodiola rosea, and Lonicera caerulea against Escherichia coli. Extracts were chemically characterized by HPLC-MS and evaluated using both standard 96-well plate microdilution and droplet-based assays. In total, 27,059 droplets were segmented in CP, classified into four growth phenotypes using a CPA random forest model, and analyzed in EF2. The droplet-based assay reached minimal inhibitory concentration (MIC) at lower extract concentrations than 96-well plate microdilution and revealed extract-specific aggregation effects: L. caerulea reduced E. coli aggregation, whereas T. officinale did not. R. rosea reduced growth in the 96-well plate microdilution assay, but high concentrations caused droplet gelation that prevented droplet-based analysis, showing that sample components must be checked for compatibility with droplet formation. These results illustrate that the EF2-centered workflow supports rapid, automated MIC estimation through unsupervised thresholding when labels are unavailable, and four-class aggregation phenotyping when a CPA model is supplied. This workflow offers flexible, scalable screening, potentially extendable beyond plant extracts to other complex sample matrices.

microbiology↗