Engineered CRISPR/Cas13d Sensing hTERT Selectively Inhibits the Progression of Bladder Cancer In Vitro

Zhuang, Cheng‐Le; Zhuang, Changshui; Zhou, Qun; Huang, Xueting; Gui, Yaoting; Lai, Yongqing; Yang, Shangqi

doi:10.3389/fmolb.2021.646412

Cited by 17 publications

(17 citation statements)

References 35 publications

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“…Unfortunately the bacterial origin of PAL likely limit therapeutic applications of this system, but it represents an interesting optogenetic tool. In addition to controlling gRNA function, riboswitches have also been used to control expression of CRISPR-Cas effector proteins: Zhuang et al used an aptazyme off-switch regulated by the cancer biomarker hTERT to control expression of Cas13d, achieving selective killing of hTERT-expressing bladder cancer cells [190].…”

Section: Regulation Of Crispr-cas Activity By Riboswitchesmentioning

confidence: 99%

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Tickner

Farzan

2021

Pharmaceuticals

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Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

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Section: Regulation Of Crispr-cas Activity By Riboswitchesmentioning

confidence: 99%

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Tickner

Farzan

2021

Pharmaceuticals

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“…Notably, some of Cas13d's most successful applications in mammalian cells have been cases in which collateral activity may synergistically align with experimental goals rather than detract from or confound them. For example, Cas13d targeting cancer-specific transcripts can induce apoptosis of bladder and pancreatic cancer cells in vitro and halt tumor progression in xenograft models (Jiang et al, 2020;Li et al, 2021a;Zhuang et al, 2021). In this context, extensive collateral activity would likely exacerbate cancer-specific toxicity by interfering with critical cellular processes.…”

Section: Discussionmentioning

confidence: 99%

Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

Kelley

Haerle

Wang

2021

Preprint

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Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.

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“…The method also employs the use of OFF-switch hTERT aptazyme incorporated into the 3′ UTR of the Cas13d, which can further inhibit the degradation of Cas13d in bladder cancer cells. Further, their study also revealed that the incorporation of reconstructed CRISPR/Cas13d sensing hTERT in bladder cancer 5637 and T24 cells was found to induce cell apoptosis and subsequently inhibit cell proliferation, migration, and invasion without affecting normal human foreskin fibroblast (HFF) cells ( Zhuang et al, 2021 ). Hence, CRISPR/Cas13d sensing hTERT approach can serve as a prominent therapy to control the tumorogenesis of bladder cancer.…”

Section: Various Applications Of Cas13d In Basic Research and Biomedi...mentioning

confidence: 99%

Cas13d: A New Molecular Scissor for Transcriptome Engineering

Gupta¹,

Ghosh²,

Chakravarti³

et al. 2022

Front. Cell Dev. Biol.

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The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes.

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Engineered CRISPR/Cas13d Sensing hTERT Selectively Inhibits the Progression of Bladder Cancer In Vitro

Cited by 17 publications

References 35 publications

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

Cas13d: A New Molecular Scissor for Transcriptome Engineering

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