Precise RNA targeting with CRISPR-Cas13d
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE284017 | GEO | 2024/12/18
REPOSITORIES: GEO
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Project description:The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, the collateral activity of LwaCas13a mildly impeded but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.
2024-01-25 | GSE254168 | GEO
Project description: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.
2021-12-23 | GSE191329 | GEO
Project description:We found that several variants with 2-4 mutations on Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains showed undiminished on-target activity but markedly reduced collateral effects. Furthermore, transcriptome-wide off-target effects and cell growth arrest induced by wild-type Cas13d were found to be absent for a Cas13d variant. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for the targeted degradation of RNAs in basic research and therapeutic applications.
2022-06-28 | GSE168246 | GEO
Project description:Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo.
2025-01-22 | GSE284120 | GEO
Project description:Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo.
2025-01-22 | GSE270530 | GEO
Project description:Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo.
2025-01-22 | GSE270529 | GEO
Project description:Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo.
2025-01-22 | GSE270528 | GEO
Project description:Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo.
2025-01-22 | GSE270527 | GEO
Project description:An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
2024-12-08 | GSE283702 | GEO
Project description:High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects
| PRJNA706673 | ENA