CRISPR undoubtedly transformed genome editing, but it left two crucial problems unsolved. Cas nucleases cut the chromosome and leave the cell to repair the break, an error-prone process that is inefficient for installing new DNA, and Cas9 is large relative to the cargo that viral vectors can carry for gene therapy. A new generation of genome editors derived from mobile genetic elements is emerging to address both issues, particularly from the bacterial transposons and insertion sequences colloquially known as ‘jumping genes’. Like CRISPR, these systems use short, programmable RNAs to locate their target sequence, but each new system offers distinct advantages. CRISPR-associated transposases (CASTs) insert whole genes at defined sites without double-stranded breaks, the compact TnpB and IscB nucleases are around a third of the size of Cas9 and fit well inside viral vectors for gene therapy delivery, IS110 bridge recombinases can insert, excise and invert DNA without scarring, and the newly discovered TIGR-Tas systems read their target with paired guide sequences for added precision. This review traces the origins of these tools in bacterial mobile genetic elements and how they edit DNA, what sets them apart from CRISPR, and their emerging uses across medicine, agriculture and microbial engineering.
Christopher Jin, J. Tree· Microbiology Australia· 0 citations
Bacterial small RNAs (sRNAs) regulate gene expression by base pairing with target mRNAs, yet transcriptome-wide interactome mapping has shown that many sRNA–mRNA interactions detected in vivo have modest or no regulatory effect using orthogonal reporter assays. The features that determine functional outcome remain poorly defined. Here, we integrated Hfq-CLASH interactome mapping with matched transcriptomic and proteomic profiling in Escherichia coli and developed an interpretable machine-learning framework to identify the determinants that distinguish functional from non-functional interactions. Using sequence, structural, thermodynamic, duplex and protein-occupancy features, transcriptomic and proteomic responses were predicted with above-chance performance, achieving AUCs of 0.78 and 0.74, respectively. Feature attribution revealed that physical pairing alone is insufficient for regulation; instead, regulatory outcome is shaped by a coordinated interplay between RNA secondary structure, thermodynamic accessibility and local protein-binding context. Target-side Hfq occupancy emerged as a positive predictor of functional regulation, whereas AR2-domain occupancy on the sRNA was associated with non-responsive interactions, suggesting that distinct ribonucleoprotein states may separate productive regulation from non-productive binding. These findings indicate that the regulatory fate of an sRNA–mRNA interaction is an emergent property of its biophysical context and protein-binding environment, rather than a direct consequence of physical pairing alone. GRAPHICAL ABSTRACT
F. Safari, Daniel G. Mediati, Saleh Alquethamy et al.· bioRxiv· 0 citations
Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogeneic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet their expression must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA that is enriched in the locus of enterocyte effacement (LEE⁺) E. coli strains and present in up to nine copies in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes. HnrS base-pairs with the hha ribosome-binding site to inhibit translation, thereby modulating Hha–H-NS repression of virulence loci including the LEE type III secretion system. Loss of HnrS alters motility, T3SS expression, and a subset of Hha-regulated genes. These findings reveal an RNA-based counter-silencing strategy encoded by prophage to relieve xenogenic silencing.
Pranita Poudyal, Brandon M. Sy, Daniel G. Mediati et al.· PLoS Pathogens· 0 citations
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