Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA612134 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11293500_1.fastq.gz | Fastqsanger.gz | |||
| SRR11293500_2.fastq.gz | Fastqsanger.gz | |||
| SRR11293501_1.fastq.gz | Fastqsanger.gz | |||
| SRR11293501_2.fastq.gz | Fastqsanger.gz | |||
| SRR11293502_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects.
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Project description:RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a “proof of principle” demonstration that CRISPR endoribonuclease can be used for programmable mRNA transcript degradation in eukaryotes. Using zebrafish as the animal model and Csm complex as the CRISPR endoribonuclease, we targeted EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We also tested Csm mediated knockdown on the endogenous tdgf1 (oep) transcript. At optimal Csm dose, we observed a penetrance of the characteristic one-eyed phenotype at greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown with no significant off-target effects in our model.
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Project description:Enhanced RNA-targeting CRISPR-Cas technology in zebrafish III
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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.
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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.
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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.
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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.
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Project description:Characteristic for type III CRISPR-Cas systems is the generation of cyclic oligoadenylate (cOA) molecules, which allosterically activate proteins carrying cognate sensory domains: CARF or SAVED. Here, we characterize a type III-B system associated set of genes from Haliangium ochraceum, containing two CBASS co-opted caspase-like proteases, SAVED-CHAT and PCaspase (Prokaryotic Caspase). Cyclic tri-AMP (cA3)- induced oligomerization of the SAVED domains of SAVED-CHAT leads to proteolytic activity of the CHAT domains which, when active, specifically cleave and activate PCaspase. In turn, activated PCaspase cleaves a multitude of proteins and leads to a strong defence mechanism in vivo in E. coli.. Together, our findings show a cascade of proteolytic activities (with conceptual similarities to eukaryotic caspases) and constitute an effective strategy to defend against mobile genetic elements.
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