Biology subjects

Aird, E. J.

Publications and source records attributed to Aird, E. J..

6 recordsLinked to original sources

bioRxiv2024

SP110 sequestration of SP100 protects against toxic filaments during innate immune signaling

Stimulation of the innate immune system by foreign RNA elicits a potent response against invading pathogens and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Employing genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we identify speckled protein 110 (SP110) as a potent negative regulator of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a death-promoting activity of another speckled protein, SP100. Both SP110 suppression and SP100 toxicity are mediated by direct interactions between the caspase activation and recruitment domains (CARDs) in each protein. SP100-induced death is mediated by homomeric CARD filaments that are disassembled by a heteromeric CARD interaction with SP110. Overexpression of SP100 is sufficient to overwhelm normal levels of SP110, leading to genotoxicity. Using cryo-EM and AlphaFold modeling, we develop and validate an atomic description of SP100 CARD filament formation and filament breaking by SP110. Genome-wide binding studies reveal that SP110 and SP100 normally associate at active promoters, but disruption of the CARD interaction releases SP100 to form toxic filaments. Overall, we uncover a novel regulatory partnership in human innate immunity that balances signal potency with cell intrinsic lethality.

immunology↗

bioRxiv2021

Split Staphylococcus aureus prime editor for AAV delivery

Prime editing brings immense promise to correct a large number of human pathogenic mutations and enact diverse edit types without introducing widespread undesired editing events. Delivery of prime editors in vivo would enable such edits to be introduced in a clinical setting. The coding sequence for prime editor, however, is too large to fit within the size-constrained adeno-associated virus (AAV) genome. Herein, we describe a split Staphylococcus aureus prime editor capable of being delivered by dual AAVs. We characterize the editing ability of plasmid-based versions of an S. aureus prime editor in vitro at a variety of loci with diverse edit types. We investigate various split prime editor architectures and alternative dimerization domains. Finally, we demonstrate the capacity of prime editor to be co-delivered by dual AAVs in vitro. While editing rates are lower than desired, this approach presents an important step to translate prime editing for in vivo delivery.

molecular biology↗

bioRxiv2020

Molecular underpinnings of ssDNA specificity by Rep HUH endonucleases and implications for HUH-tag multiplexing and engineering

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

biophysics↗

bioRxiv2019

An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)

Molecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

cell biology↗

bioRxiv2018

Harnessing Notch signaling to decode mechanisms of proteolytic regulation in diverse cell-surface receptors

Proteolysis of transmembrane receptors is a critical cellular communication mechanism dysregulated in many diseases, yet decoding proteolytic regulation mechanisms of the estimated 400 receptors shed from the cell surface has been hindered by difficulties in controlling stimuli and unknown fates of cleavage products. Notch proteolytic regulation is a notable exception, where decades of study have revealed that intercellular forces drive exposure of a cryptic protease site within a juxtamembrane \"proteolytic switch\" domain to activate transcriptional programs inside the cell. Thus, we created a Synthetic Notch Assay for Proteolytic Switches (SNAPS) that exploits the modularity and unequivocal input/response of Notch proteolysis to screen surface receptors for other putative proteolytic switches. Here, we identify several new proteolytic switches among receptors with structural homology to Notch. We demonstrate that SNAPS can detect shedding in chimeras of diverse cell surface receptors, leading to new, testable hypotheses. Finally, we establish that the assay can be used to measure modulation of proteolysis by potential therapeutics.

systems biology↗

bioRxiv2017

Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome where the Cas9 nuclease then induces a double stranded break (DSB)1,2. The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB3,4. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded donor oligonucleotide (ssODN) to the Cas9/guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease5, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to an 8-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci, and is more pronounced at low RNP concentrations.

molecular biology↗