Biology subjects

Varble, A.

Publications and source records attributed to Varble, A..

3 recordsLinked to original sources

bioRxiv2026

A Staphylococcus phage recombination cassette that enables recombineering across distant bacteria

Modifying bacterial genomes with phage recombinases, a process termed recombineering, has enabled precise genome editing, but its reach is limited by host specificity: each new organism generally requires its own recombinase to be identified and optimized. Using known phage recombination genes as synteny anchors, we mined Staphylococcus genomes and recovered a diverse collection of phage recombination cassettes, spanning the RAD52-like annealase superfamily (Sak, Erf) and the RAD51/RecA-like Sak4 family. Functional testing of representatives in S. aureus identified several highly efficient systems that support recombineering. We engineered the most efficient of these into a single-plasmid tool that yields markerless editing in S. aureus in four days. Notably, where other recombineering systems rely on host-specific factors for nuclease evasion and ssDNA loading, this cassette encodes both activities itself: a predicted broad-spectrum nuclease inhibitor and a dedicated single-stranded DNA-binding protein. We therefore tested the cassette across phylogenetically distant hosts, and observed recombination in Streptococcus pyogenes, Enterococcus faecium, Francisella novicida, and Escherichia coli. These results identify a self-sufficient class of phage recombination systems and provide a portable tool for

microbiology↗

bioRxiv2024

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacteriums poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based sgRNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just two days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ-inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. Significance statementWhile GAS remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS, and together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

microbiology↗

bioRxiv2020

Integration of prophages into CRISPR loci remodels viral immunity in Streptococcus pyogenes

CRISPR loci are composed of short DNA repeats separated by sequences that match the genomes of phages and plasmids, known as spacers. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defense, phages can produce small proteins that inhibit these nucleases. Here we demonstrate that the {Phi}AP1.1 temperate phage utilizes an alternate approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection this phage expresses a canonical anti-CRISPR, AcrIIA23 that prevents Cas9 function, allowing {Phi}AP1.1 to integrate into the direct repeats of the CRISPR locus and neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transfer of spacers, enabling a complex modulation of the type II-A CRISPR immune response.

microbiology↗