Stapleton, J. A. | Search bioRxiv

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2025

Optimizing in-vitro Transcribed CRISPR-Cas9 single-guide RNA Libraries for Improved Uniformity and Affordability

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer, immediately downstream of the T7 promoter, as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.

molecular biology↗