Biology subjects

Robertson, L. J.

Publications and source records attributed to Robertson, L. J..

3 recordsLinked to original sources

bioRxiv2025

The LAST mile: Evaluating genetic biocontrol as a supplemental tool for eradicating invasive rodents on islands

Invasive rodents cause severe ecosystem degradation on islands and can be challenging to eradicate. Current best-practices rely heavily on the application of toxic oral baits and have led to successful eradications and remarkable recoveries of native flora and fauna. Yet this single method is not universally applicable. Reliance on a single method limits further eradication success in situations where toxicants cannot be used or remnant populations persist after application. Genetic biocontrol offers a suite of new solutions to potentially improve outcomes in the critical "last mile" of eradication. These approaches involve the release of genetically modified individuals of the target species to reduce population fitness over time. These include self-limiting approaches which require multiple releases and self-sustaining mechanisms (i.e., select gene-drive systems) which could theoretically collapse populations after a single release. While gene drive systems have received significant attention, their development in vertebrates remains technically challenging, and their ecological and regulatory implications are still in active debate. In contrast, some non-drive genetic biocontrol approaches, such as Y-linked editors, fsRIDL, and Gravid Lethal, offer self-limiting alternatives that may be more immediately deployable. These approaches could be used to supplement toxicant-based methods and may also have reduced environmental risks and regulatory barriers. To evaluate the potential of these tools, we developed an individual-based model simulating the eradication of house mice (Mus musculus) on a 125ha island with an initial population of 11,000 individuals. We tested various combinations of genetic biocontrol release and effort strategies to understand tradeoffs between required effort and uncertainty for achieving successful eradication. Under certain effort strategies, we found that the Gravid Lethal approach performed the best, achieving eradication in less than 2.3 years with intensive release effort and monitoring. Our findings suggest that integrating non-drive genetic biocontrol into adaptive management frameworks could enhance the effectiveness and feasibility of rodent eradication programs. These tools are not replacements for toxicants but may serve as critical supplements--particularly in the "last mile" of eradication. Further stakeholder engagement is needed to assess the ecological, ethical, and logistical dimensions of deploying these technologies in real-world settings.

ecology↗

bioRxiv2024

Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9- and SaCas9-mediated Gene Editing

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes.

molecular biology↗

bioRxiv2024

Functional analysis of the epilepsy gene Pcdh19 using a novel GFP-reporter mouse model

PCDH19 is a cell adhesion molecule belonging to the delta2-protocadherin subfamily that plays a critical role in brain development, neuronal migration, synaptic organisation, and neural circuit formation. Mutations in PCDH19 cause PCDH19-clustering epilepsy, an infantile-onset disorder characterized by seizures and intellectual disabilities. Despite the increasing development of constitutive cellular and murine models to investigate the effects of Pcdh19 knockout on cell-cell interactions and cellular function, the spatiotemporal consequences of its loss remain poorly understood. To address this gap, we generated and validated a novel conditional Pcdh19 knockout mouse model incorporating a GFP reporter (Pcdh19-cKO-GFP), enabling cell type-specific and temporally controlled gene deletion and direct visualization of recombination events. Using a neuronal Syn1-Cre driver, we demonstrate that targeted deletion of Pcdh19 in neurons results in altered hippocampal neurogenesis and mouse behaviour. We further demonstrate the versatility of this model using a doxycycline-inducible Cre system, enabling temporally controlled deletion of Pcdh19 and the modelling of disease-relevant phenotypes. Finally, we validate adeno-associated viral (AAV) vector-mediated recombination as a strategy for precise postnatal manipulation of Pcdh19 expression. Collectively, this Pcdh19-cKO-GFP model provides a powerful and flexible genetic tool to interrogate the cell type specific and temporary regulated functions of PCDH19 in the developing and postnatal brain under physiological and disease conditions.

neuroscience↗