da Silva, J. F. | bioRxiv Science

bioRxiv2023

Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the "click"-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether "click DNA" (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

bioengineering↗

bioRxiv2023

BKCa nitrosylation is associated with cerebral microvascular dysfunction in female 5x-FAD mice

BackgroundCerebral microvascular dysfunction and nitro-oxidative stress are present in patients with Alzheimers disease (AD) and may contribute to disease progression and severity. Large conductance Ca2+-activated K+ channels (BKCa) play an essential role in vasodilatory responses and maintenance of myogenic tone in resistance arteries. BKCa impairment can lead to microvascular dysfunction and hemodynamic deficits in the brain. We hypothesized that reduced BKCa function in cerebral arteries mediates microvascular and neurovascular responses in the 5x-FAD model of AD. MethodsBKCa activity in the cerebral microcirculation was assessed by patch clamp electrophysiology and pressure myography, in situ Ca2+ sparks by spinning disk confocal microscopy, hemodynamics by laser speckle contrast imaging. Molecular and biochemical analyses were conducted by affinity-purification assays, qPCR, Western blots and immunofluorescence. ResultsWe observed that pial arteries from 5-6 months-old male and female 5x-FAD mice exhibited a hyper-contractile phenotype than wild-type (WT) littermates, which was linked to lower vascular BKCa activity and reduced open probability. In males, BKCa dysfunction is likely a consequence of an observed lower expression of the pore-forming subunit BK and blunted frequency of Ca2+ sparks, which are required for BKCa activity. However, in females, impaired BKCa function is, in part, a consequence of reversible nitro-oxidative changes in the BK subunit, which reduces its open probability and regulation of vascular tone. We further show that BKCa function is involved in neurovascular coupling in mice, and its dysfunction is linked to neurovascular dysfunction in the model. ConclusionThese data highlight the central role played by BKCa in cerebral microvascular and neurovascular regulation, as well as sex-dependent mechanisms underlying its dysfunction in a mouse model of AD.

physiology↗