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Abstract Background Invasive management strategies range from preventing new invasive species incursions to eliminating established populations, with all requiring effective monitoring to guide action. The use of DNA sampled from the environment (eDNA) is one such tool that provides the ability to surveille and monitor target invasive species through passive sampling. Technology being developed to eliminate invasive species includes genetic biocontrol in the form of gene drive. This approach would drive a trait through a population and could be used to eliminate or modify a target population. Once a gene drive organism is released into a population then monitoring changes in density of the target species and the spread of the drive in the population would be critical. Results In this paper, we use invasive Mus musculus as a model for development of an eDNA assay that detects wild-type M. musculus and gene drive M. musculus. We demonstrate successful development of an assay where environmental samples could be used to detect wild-type invasive M. musculus and the relative density of wild-type to gene drive M. musculus. Conclusions The development of a method that detects both wild-type M. musculus and a gene drive M. musculus (t CRISPR) from environmental samples expands the utility of environmental DNA. This method provides a tool that can immediately be deployed for invasive wild M. musculus management across the world. This is a proof-of-concept that a genetic biocontrol construct could be monitored using environmental samples.

Abstract Background The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. Results To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. Conclusions Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

Abstract Pathogenic variants in the gene encoding the small GTPase Ras analogue in Brain 39b (RAB39B) are associated with early-onset parkinsonism. In this study we investigated the expression and localization of RAB39B (RNA and protein) in mouse brain tissue to gain a better understanding of its normal physiological function(s) and role in disease. We developed novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, and performed real-time PCR and western blot analysis on whole brain lysates. To determine the spatial localization of Rab39b RNA and protein, we performed in-situ hybridization and immunohistochemistry on fresh frozen and fixed brain tissue. Our results show that RAB39B is localized throughout the cortex, hippocampus and substantia nigra of mice throughout postnatal life. We found high levels of RAB39B within MAP2 positive cortical and hippocampal neurons, and TH positive dopaminergic neurons in the substantia nigra pars compacta. Our studies support and extend current knowledge of the localization of RAB39B. We validate RAB39B as a neuron-enriched protein and demonstrate that it is present throughout the mouse cortex and hippocampus. Further, we observe high levels in the substantia nigra pars compacta, the brain region most affected in Parkinson’s disease pathology. The distribution of Rab39b is consistent with human disease associations with parkinsonism and cognitive impairment. We also describe and validate novel resources, including monoclonal antibodies directed against RAB39B and mice with Rab39b knockout, both of which are valuable tools for future studies of the molecular function of RAB39B.