Biology subjects

Gierus, L.

Publications and source records attributed to Gierus, L..

5 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↗

bioRxiv2023

Investigating the potential of X shredding for mouse genetic biocontrol

CRISPR-Cas9 technology has facilitated development of strategies that can potentially provide more humane and effective methods to control invasive vertebrate species, such as mice. One promising strategy is X chromosome shredding which aims to bias offspring towards males, resulting in a gradual and unsustainable decline of females. This method has been explored in insects with encouraging results. Here, we investigated this strategy in Mus musculus by targeting repeat DNA sequences on the X chromosome with the aim of inducing sufficient DNA damage to specifically eliminate X chromosome-bearing sperm during gametogenesis. We tested three different guide RNAs (gRNAs) targeting different repeats on the X chromosome, together with three male germline-specific promoters for inducing Cas9 expression at different stages of spermatogenesis. A modest bias towards mature Y-bearing sperm was detected in some transgenic males, although this did not translate into significant male-biasing of offspring. Instead, cleavage of the X-chromosome during meiosis typically resulted in a spermatogenic block, manifest as small testes volume, empty tubules, low sperm concentration, and sub/infertility. Our study highlights the importance of controlling the timing of CRISPR-Cas9 activity during mammalian spermatogenesis and the sensitivity of spermatocytes to X chromosome disruption.

molecular biology↗

bioRxiv2022

Leveraging a natural murine meiotic drive to suppress invasive populations

Invasive rodents, including house mice, are a major cause of environmental damage and biodiversity loss, particularly in island ecosystems. Eradication can be achieved through the distribution of rodenticide, but this approach is expensive to apply at scale, can have negative impacts (e.g. on non-target species, or through contamination), has animal ethics concerns, and has restrictions on where it can be used. Gene drives, which exhibit biased inheritance, have been proposed as a next generation strategy to control invasive alien pests and disease vectors. However, synthetic gene drives including CRISPR homing drives have proven to be technically challenging to develop in mice. The t haplotype is a naturally-occurring segregation distortion locus with highly biased transmission from heterozygous males. Here we propose a novel gene drive strategy for population suppression, tCRISPR, that leverages t haplotype bias and an embedded SpCas9/gRNA transgene to spread inactivating mutations in a haplosufficient female fertility gene. Using spatially explicit individual-based in silico modelling, we show that polyandry, sperm competition, dispersal, and transmission bias are critical factors for tCRISPR-mediated population suppression. Modelling of realistic parameter values indicates that tCRISPR can eradicate an island population of 200,000 mice while the unmodified t haplotype fails under the same conditions. We also demonstrate feasibility of this approach by engineering tCRISPR mice in a safe split drive format. tCRISPR mice exhibit high transmission of the modified t haplotype, and efficient generation and transmission of inactivating mutations in a recessive female fertility gene, crucially, at levels for which the modelling predicts that population eradication can occur. This is the first example of a feasible gene drive system for invasive alien rodent population control.

genetics↗

bioRxiv2021

Optimized nickase- and nuclease-based prime editing in human and mouse cells

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. Currently, the delivery of PE components to mammalian cell lines requires multiple plasmid vectors. To overcome this limitation, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency. In summary, PE-nuclease and the PEA1 plasmids provide new tools to generate intended edits with high efficiency.

molecular biology↗