Biology subjects

Child, M. A.

Publications and source records attributed to Child, M. A..

7 recordsLinked to original sources

bioRxiv2026

Using Polycistronic gRNAs and a tRNA Cleavage System in CRISPR-Based Genome Editing of Toxoplasma gondii

CRISPR/Cas9 is widely used for genetic manipulation in Toxoplasma gondii, but optimizing gRNA expression and multiplexed editing remains challenging. Here, we evaluated alternative promoters, gRNA scaffolds, and a tRNA-based polycistronic system to improve editing efficiency and reduce synthetic DNA costs. Four constructs targeting the SAG1 locus were used to compare the U6 and H1 promoters, as well as standard and modified gRNA scaffolds. U6-driven constructs supported robust gRNA-targeted Cas9 activity, whereas H1-driven constructs failed to produce detectable phenotypes. The alternative gRNA scaffold, modified by a number of base changes and insertions, increased knockout efficiency compared to the standard scaffold. To simplify dual gRNA-based methods, we establish a tRNA-based polycistronic system to simultaneously target two independent loci. Targeting the SAG1 and UPRT locus for proof-of-concept, all U6-driven polycistronic constructs conferred efficient SAG1 knockout and 5-fluorodeoxyuridine resistance, whereas H1-driven constructs exhibited position-dependent activity, with only the 3' gRNA being functional. These results demonstrate that U6-driven, tRNA-based polycistronic gRNA expression enables reliable multiplexed genome editing in T. gondii, with the minimal H1 promoter being insufficient for consistent multi-gRNA activity. This optimized system provides a scalable framework for dual-gRNA CRISPR strategies in this organism.

molecular biology↗

bioRxiv2026

CORe Designer: a CRISPR design tool for proteome engineering

Precision proteome engineering requires tools that leverage CRISPR technologies, yet most current design platforms remain gene-centric. To bridge this gap, we introduce CORe Designer. Featuring an intuitive graphical user interface, CORe Designer enables researchers to design guide RNAs and homology-directed repair templates in a protein-centric manner. By automating these workflows, CORe Designer enables the design of targeted mutagenesis experiments for precision proteome engineering to be completed in minutes.

bioengineering↗

bioRxiv2024

Highly conserved brain vascular receptor ALPL mediates transport of engineered viral vectors across the blood-brain barrier

Delivery of systemically administered therapeutics to the central nervous system (CNS) is restricted by the blood-brain barrier (BBB). Bioengineered Adeno-Associated Virus (AAV) capsids have been shown to penetrate the BBB with great efficacy in mouse and non-human primate models, but their translational potential is often limited by species selectivity and undefined mechanisms of action. Here, we apply our RNA-guided TRACER AAV capsid evolution platform to generate VCAP-102, an AAV9 variant with markedly increased brain tropism following intravenous delivery in both rodents and primates. VCAP-102 demonstrates a similar CNS tropism in cynomolgus macaque, african green monkey, marmoset and mouse, showing 20- to 400-fold increased transgene expression across multiple brain regions relative to AAV9. We demonstrate that the enhanced CNS tropism of VCAP-102 results from direct interaction with alkaline phosphatase (ALPL), a highly conserved membrane-associated protein expressed on the brain vasculature. VCAP-102 interacts with human, primate and murine ALPL isoforms, and ectopic expression of ALPL is sufficient to initiate receptor-mediated transcytosis of VCAP-102 in an in vitro transwell model. Our work identifies VCAP-102 as a cross-species CNS gene delivery vector with a strong potential for clinical translation and establishes ALPL as a brain delivery shuttle capable of efficient BBB transport to maximize CNS delivery of biotherapeutics.

neuroscience↗

bioRxiv2021

An extracellular redox signal triggers calcium release and impacts the asexual development of Toxoplasma gondii

The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as Toxoplasma gondii. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H2O2 to human fibroblasts infected with T. gondii triggers a Ca2+ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H2O2 is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in T. gondii, evidencing a link between extracellular redox and intracellular Ca2+ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.

microbiology↗

bioRxiv2021

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

microbiology↗

bioRxiv2020

Cellular barcoding of Toxoplasma reveals permissive host brain colonization

Molecular barcoding techniques have emerged as powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been extended to protozoan parasites, which have unique genomic structures and virulence strategies compared to viral and bacterial pathogens. Here, we present a versatile CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. The murine brain is an important transmission niche for T. gondii, and brain persistence is a clinically untreatable feature of infection. The blood-brain barrier is expected to physically restrict parasite colonization of this niche, resulting in a selection bottleneck. Using libraries of barcoded T. gondii we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one-month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes that represented a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. Together, these data establish the first, robust molecular barcoding approach for protozoa and evidence that the blood-brain barrier does not represent a major bottleneck to colonization by T. gondii.

microbiology↗

bioRxiv2020

CRISPR-TAPE: protein-centric CRISPR guide design for targeted proteome engineering

Rational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. To address this, we have developed CRISPR-TAPE, a protein-centric gRNA design algorithm that allows users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time consuming post-hoc curation, simplifying gRNA outputs, and reducing CPU times.

bioinformatics↗