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Published on: May 23, 2016
Targeted gene silencing in the nervous system with CRISPR-Cas13
Jackson E Powell1, Colin K W Lim1, Ramya Krishnan1
1Department of Bioengineering, University of Illinois, Urbana, IL 61801, USA.
Abstract:
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.
Insights
RfxCas13d efficiently silences neurodegeneration-linked genes in the mouse nervous system. This CRISPR-based gene silencing platform shows promise for treating disorders like amyotrophic lateral sclerosis and Huntington's disease.
Area of Science:
- Molecular Biology
- Neuroscience
- Gene Editing
Background:
- Cas13 nucleases are RNA-targeting CRISPR effectors for gene silencing.
- RfxCas13d is a Cas13 ortholog with advantageous properties.
- Neurodegenerative diseases are linked to specific gene expressions.
Purpose of the Study:
- To evaluate RfxCas13d's efficacy in silencing neurodegeneration-associated genes in the mammalian nervous system.
- To demonstrate RfxCas13d delivery and gene knockdown in the mouse spinal cord and brain.
- To assess RfxCas13d's therapeutic potential in mouse models of neurological disorders.
Main Methods:
- Adeno-associated virus (AAV) vector delivery of RfxCas13d variants targeting SOD1 and HTT genes.
- Intrathecal and intrastriatal administration in mouse models.
- Quantification of target mRNA and protein levels (SOD1 and HTT).
- Assessment of disease outcomes in a mouse model of amyotrophic lateral sclerosis.
Main Results:
- RfxCas13d targeting superoxide dismutase 1 (SOD1) reduced SOD1 mRNA and protein by over 50% in the mouse spinal cord.
- This SOD1 knockdown improved outcomes in an amyotrophic lateral sclerosis mouse model.
- RfxCas13d targeting huntingtin (HTT) reduced HTT protein by approximately 50% in the mouse brain.
- Demonstrated successful delivery and gene silencing in both the spinal cord and brain.
Conclusions:
- RfxCas13d is a versatile CRISPR-based platform for gene knockdown in the nervous system.
- RfxCas13d shows potential for therapeutic applications in neurodegenerative diseases.
- The study establishes RfxCas13d as a viable tool for targeting RNA in the central nervous system.
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