Biology subjects

Cassidy, A.

Publications and source records attributed to Cassidy, A..

4 recordsLinked to original sources

bioRxiv2025

eSPLIT and iSWAP: CRISPR-Mediated Conditional Allele Engineering with Short Artificial Introns

Engineering conditional alleles remains a major challenge in functional genomics. Short Artificial Introns (SAIs) have emerged as powerful tools to simplify allele design and characterization, yet practical guidelines for their implementation remain limited. Here, we describe two streamlined strategies, eSPLIT (exon split) and iSWAP (intron swap), that enable efficient generation of conditional alleles using SAIs. In eSPLIT, a compact cassette containing essential intronic elements flanked by loxP sites is integrated within an exon, whereas in iSWAP, a native intron is replaced with an SAI. In the absence of Cre recombinase, the SAI is recognized as an intron and removed by the splicing machinery, allowing normal gene expression. Following Cre-mediated recombination, excision of critical intronic sequences disrupts splicing, leaving residual sequences, including stop codons in all reading frames, thereby causing premature translation termination and gene inactivation. We optimized these approaches by generating a series 18 SCYL1 alleles in human cells, validated their general applicability in 5 mouse models across multiple genes and further extended the approach to the FLP-FRT system in vivo. By defining practical rules for SAI placement, we establish a robust and scalable framework for engineering conditional alleles, with broad utility in functional genomics and disease modeling. SHORT 100 - WORD ABSTRACTWe introduce eSPLIT and iSWAP, two streamlined strategies for generating conditional alleles using a short artificial intron (SAI). eSPLIT integrates a loxP-flanked SAI within an exon, while iSWAP replaces a native intron. Cre-mediated excision disrupts SAI splicing, leading to gene inactivation. Optimized using SCYL1 alleles in human cells and validated in mouse models across multiple genes, these strategies provide a simple and efficient approach for engineering conditional alleles. By outlining design rules for SAI placement, we provide a robust and scalable strategy for functional genomics and disease modeling.

genomics↗

bioRxiv2021

Divergent early antibody responses define COVID-19 disease trajectories

A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-Fc{gamma}R interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-Fc{gamma}R interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2. One Sentence SummaryDivergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo.

immunology↗

bioRxiv2020

Genome-scale functional profiling of cell cycle controls in African trypanosomes

Trypanosomatids, which include major pathogens of humans and livestock, are divergent eukaryotes for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle regulators in bloodstream form Trypanosoma brucei. We induced massive parallel knockdown and sorted the perturbed population into cell cycle stages using flow cytometry. RNAi-targets were deep-sequenced from each stage and cell cycle profiles were digitally reconstructed at a genomic scale. We identify hundreds of proteins that impact cell cycle progression; glycolytic enzymes required for G1S progression, DNA replication factors, mitosis regulators, proteasome and kinetochore complex components required for G2M progression, flagellar and cytoskeletal components required for cytokinesis, mRNA-binding factors, protein kinases and many previously uncharacterised proteins. The outputs facilitate functional annotation and drug-target prioritisation and provide comprehensive functional genomic evidence for the machineries, pathways and regulators that coordinate progression through the trypanosome cell cycle. The data can be searched and browsed using an interactive, open access, online data visualization tool (https://tryp-cycle.onrender.com).

genomics↗

bioRxiv2020

Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. Ser-Arg Repeat Protein Kinase (SRPK) is one such conserved eukaryotic kinase, which controls mRNA splicing. Surprisingly, we show that SRPK has acquired a novel function in regulating a neurodevelopmental ubiquitin signalling pathway. In mammalian embryonic stem cells, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of transcription factor substrates, thereby acting to restrain a neural gene expression programme that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signalling that ensures correct regulation of neurodevelopmental gene expression.

developmental biology↗