ORCID Profile
0000-0003-2163-0514
Current Organisation
University of Adelaide
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Biochemistry and cell biology | Synthetic biology |
Publisher: Public Library of Science (PLoS)
Date: 05-11-2021
DOI: 10.1371/JOURNAL.PONE.0258538
Abstract: Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin ( Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo . We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans -chromosomal interaction of the Nes enhancer and promoter.
Publisher: Mary Ann Liebert Inc
Date: 10-2020
Publisher: Wiley
Date: 31-07-2023
DOI: 10.1002/WSBM.1580
Abstract: CRISPR gene-editing technology creates precise and permanent modifications to DNA. It has significantly advanced our ability to generate animal disease models for use in biomedical research and also has potential to revolutionize the treatment of genetic disorders. Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disease that could potentially benefit from the development of CRISPR therapy. It is commonly associated with mutations that disrupt the reading frame of the DMD gene that encodes dystrophin, an essential scaffolding protein that stabilizes striated muscles and protects them from contractile-induced damage. CRISPR enables the rapid generation of various animal models harboring mutations that closely simulates the wide variety of mutations observed in DMD patients. These models provide a platform for the testing of sequence-specific interventions like CRISPR therapy that aim to reframe or skip DMD mutations to restore functional dystrophin expression. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics.
Publisher: Oxford University Press (OUP)
Date: 07-2017
DOI: 10.1534/GENETICS.117.202549
Abstract: Gene duplication provides spare genetic material that evolution can craft into new functions. Sox2 and Sox3 are evolutionarily related genes with overlapping and unique sites of expression during embryogenesis. It is currently unclear whether SOX2 and SOX3 have identical or different functions. Here, we use CRISPR/Cas9-assisted mutagenesis to perform a gene-swap, replacing the Sox3 ORF with the Sox2 ORF to investigate their functional equivalence in the brain and testes. We show that increased expression of SOX2 can functionally replace SOX3 in the development of the infundibular recess/ventral diencephalon, and largely rescues pituitary gland defects that occur in Sox3 null mice. We also show that ectopic expression of SOX2 in the testes functionally rescues the spermatogenic defect of Sox3 null mice, and restores gene expression to near normal levels. Together, these in vivo data provide strong evidence that SOX2 and SOX3 proteins are functionally equivalent.
Publisher: American Society for Clinical Investigation
Date: 08-12-2022
DOI: 10.1172/JCI.INSIGHT.146090
Abstract: Developmental and epileptic encephalopathies (DEE) are characterized by pharmacoresistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared to mice treated with a control ASO (non-hybridizing sequence). ASO administration at neonatal age was also well-tolerated and effective in controlling seizures and extending the lifespan of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.
Publisher: Public Library of Science (PLoS)
Date: 06-12-2017
Publisher: eLife Sciences Publications, Ltd
Date: 15-02-2019
DOI: 10.7554/ELIFE.41873
Abstract: Self-replicating gene drives that modify sex ratios or infer a fitness cost could be used to control populations of invasive alien species. The targeted deletion of Y sex chromosomes using CRISPR technology offers a new approach for sex bias that could be incorporated within gene-drive designs. We introduce a novel gene-drive strategy termed Y-CHromosome deletion using Orthogonal Programmable Endonucleases (Y-CHOPE), incorporating a programmable endonuclease that ‘shreds’ the Y chromosome, thereby converting XY males into fertile XO females. Firstly, we demonstrate that the CRISPR/Cas12a system can eliminate the Y chromosome in embryonic stem cells with high efficiency (c. 90%). Next, using stochastic, in idual-based models of a pest mouse population, we show that a Y-shredding drive that progressively depletes the pool of XY males could effect population eradication through mate limitation. Our molecular and modeling data suggest that a Y-CHOPE gene drive could be a viable tool for vertebrate pest control.
Publisher: Mary Ann Liebert Inc
Date: 12-2018
Abstract: The RNA-guided endonuclease CRISPR-Cas system from
Publisher: eLife Sciences Publications, Ltd
Date: 25-01-2019
Publisher: Elsevier BV
Date: 08-2017
Publisher: Springer Science and Business Media LLC
Date: 08-2018
Publisher: Springer Science and Business Media LLC
Date: 30-06-2017
DOI: 10.1038/S41598-017-04138-1
Abstract: Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin −/− mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.
Start Date: 2023
End Date: 12-2025
Amount: $458,238.00
Funder: Australian Research Council
View Funded Activity