Publications by authors named "Tristan X McCallister"

2 Publications

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Targeted gene silencing in the nervous system with CRISPR-Cas13.

Sci Adv 2022 01 19;8(3):eabk2485. Epub 2022 Jan 19.

Department of Bioengineering, University of Illinois, Urbana, IL 61801, USA.

Cas13 nucleases are a class of programmable RNA-targeting CRISPR effector proteins that are capable of silencing target gene expression in mammalian cells. Here, we demonstrate that RfxCas13d, a Cas13 ortholog with favorable characteristics to other family members, can be delivered to the mouse spinal cord and brain to silence neurodegeneration-associated genes. Intrathecally delivering an adeno-associated virus vector encoding an RfxCas13d variant programmed to target superoxide dismutase 1 (SOD1), a protein whose mutation can cause amyotrophic lateral sclerosis, reduced SOD1 mRNA and protein in the spinal cord by >50% and improved outcomes in a mouse model of the disorder. We further show that intrastriatally delivering an RfxCas13d variant programmed to target huntingtin (HTT), a protein whose mutation is causative for Huntington’s disease, led to a ~50% reduction in HTT protein in the mouse brain. Our results establish RfxCas13d as a versatile platform for knocking down gene expression in the nervous system.
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January 2022

Beta Human Papillomavirus 8E6 Attenuates LATS Phosphorylation after Failed Cytokinesis.

J Virol 2020 06 1;94(12). Epub 2020 Jun 1.

Division of Biology, Kansas State University, Manhattan, Kansas, USA

Beta genus human papillomaviruses (β-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, β-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, β-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how β-HPV E6 causes this change in cell signaling. An screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including proproliferative genes associated with HP inactivation. β-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B [H2CB]) to induce failed cytokinesis. This system allowed us to show that β-HPV 8E6 reduced activation of large tumor suppressor kinase (LATS), an HP kinase. LATS is required for p53 accumulation following failed cytokinesis. These phenotypes were dependent on β-HPV 8E6 destabilizing p300 and did not completely attenuate the HP. It did not alter H2CB-induced nuclear exclusion of the transcription factor YAP. β-HPV 8E6 also did not decrease HP activation in cells grown to a high density. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge, this marks the first time that a β-HPV E6 protein has been shown to hinder HP signaling. β-HPVs contribute to cSCC development in immunocompromised populations. However, it is unclear if these common cutaneous viruses are tumorigenic in the general population. Thus, a more thorough investigation of β-HPV biology is warranted. If β-HPV infections do promote cSCCs, they are hypothesized to destabilize the cellular genome. data support this idea by demonstrating the ability of the β-HPV E6 protein to disrupt DNA repair signaling events following UV exposure. We show that β-HPV E6 more broadly impairs cellular signaling, indicating that the viral protein dysregulates the HP. The HP protects genome fidelity by regulating cell growth and apoptosis in response to a myriad of deleterious stimuli, including failed cytokinesis. After failed cytokinesis, β-HPV 8E6 attenuates phosphorylation of the HP kinase (LATS). This decreases some, but not all, HP signaling events. Notably, β-HPV 8E6 does not limit senescence associated with failed cytokinesis.
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June 2020