CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration

Zhou, Chunmei; Hu, Xin; Tang, Cheng; Liu, Wenjia; Wang, Shaoran; Zhou, Yingsi; Zhao, Qingjun; Bo, Qiyu; Shi, Linqi; Sun, Xiaodong; Zhou, Haibo; Yang, Hui

doi:10.1093/nsr/nwaa033

Cited by 46 publications

(37 citation statements)

References 17 publications

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“…To date, several different Cas13 subtypes have been identified and repurposed to silence target gene expression in mammalian cells ( 5 , 8 , 10 , 11 ). Among these is the Cas13d nuclease from Ruminococcus flavefaciens XPD3002 (RfxCas13d) ( 5 ), a Cas13 ortholog that has favorable targeting characteristics to other family members ( 5 , 12 , 13 ) and is small enough to fit within a single adeno-associated virus (AAV) vector with a crRNA expression cassette for in vivo gene transfer ( 14 , 15 ). Given these features, we sought to determine whether RfxCas13d could suppress gene function in vivo.…”

Section: Introductionmentioning

confidence: 99%

Targeted gene silencing in the nervous system with CRISPR-Cas13

et al. 2022

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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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Section: Introductionmentioning

confidence: 99%

Targeted gene silencing in the nervous system with CRISPR-Cas13

et al. 2022

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“…Among such systems, CasRx was recently found to be advantageous, mostly because it is more efficient and has more robust activation during RNA-guided RNA cleavage in mammalian cells in vitro (Konermann et al, 2018;Yan et al, 2018). CasRx is a type VI-D effector (Cas13d) with a small size that can be packaged into AAVs, and thus has great potential for translational medicine (Konermann et al, 2018;Yan et al, 2018;Zhou et al, 2020). In this study, we set out to explore the feasibility of using the CasRx system for the targeted silencing of metabolic genes.…”

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confidence: 99%

Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

Peng

Huang

et al. 2020

Protein Cell

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RNA knockdown in vivo carries significant potential for disease modeling and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro (Konermann et al., 2018). Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells (Konermann et al., 2018). However, the RNA-targeting activity of Cas13d still needed to be verified in vivo. In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pc-sk9 and lncLstr, in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly downregulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver.

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“…Notably, the therapeutic potential of CRISPR/CasRx was demonstrated in mouse models of neovascular age-related macular degeneration (nAMD) using AAV vectors. Delivery of the CRISPR/CasRx system successfully suppressed the mRNA of vascular endothelial growth factor (VEGF), the key factor for pathogenic ocular angiogenesis, and also subsequently showed a reduction in the area of choroidal neovascularization (CNV), the hallmark of nAMD (Zhou et al, 2020). These studies indicate the therapeutic potential for the CRISPR/CasRx system.…”

Section: Introductionmentioning

confidence: 73%

“…With RNA-targeting systems emerging as potential therapeutic models for the treatment of ocular disease (Zhou et al, 2020;Kantor et al, 2021;Nguyen et al, 2021), a stepwise manual would be instrumental for subsequent in vivo studies. In addition, Cas13 systems have been shown potent against RNA viruses, including SARS-COV-2.…”

Section: Discussionmentioning

confidence: 99%

Methods for in vitro CRISPR/CasRx-Mediated RNA Editing

Chuang

Wang

Kumar

et al. 2021

Front. Cell Dev. Biol.

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Specific changes in the genome have been accomplished by the revolutionary gene-editing tool known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The advent of programmable RNA editing CRISPR/Cas nucleases has made this gene-editing tool safer and more precise. Specifically, CasRx, a family member of the Cas13d family, has shown great therapeutic potential. Here, we describe the in vitro methods of utilizing this powerful RNA editing platform and determine the RNA editing efficiencies for CasRx with different forms of guide RNAs (also known as gRNA or sgRNA).

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CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration

Cited by 46 publications

References 17 publications

Targeted gene silencing in the nervous system with CRISPR-Cas13

Targeted gene silencing in the nervous system with CRISPR-Cas13

Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

Methods for in vitro CRISPR/CasRx-Mediated RNA Editing

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