Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos

Hernandez-Huertas, Luis; Kushawah, Gopal; Díaz‐Moscoso, Alejandro; Tomás-Gallardo, Laura; Moreno-Sánchez, Ismael; Pescador, Gabriel da Silva; Bazzini, Ariel; Moreno-Mateos, Miguel A.

doi:10.1016/j.xpro.2021.101058

Cited by 9 publications

(14 citation statements)

References 9 publications

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“…For example, CRISPR-Cas13d mRNA knock-down of Nanog in zebrafish embryos recapitulates the molecular and developmental phenotypes of morpholinos targeting Nanog [ 3 ]. So, to dissect how loss of Nanog affects the hourly grouped transcriptional activation events, SLAM-seq might be coupled with CRISPR-Cas13d system [ 57 ]. In sum, our hourly grouped transcriptional events comprise a useful parameter to access how factors affect timely regulated zebrafish ZGA.…”

Section: Discussionmentioning

confidence: 99%

miR-430 regulates zygotic mRNA during zebrafish embryogenesis

Baia Amaral,

Egidy,

Perera

et al. 2024

Genome Biol

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Background Early embryonic developmental programs are guided by the coordinated interplay between maternally inherited and zygotically manufactured RNAs and proteins. Although these processes happen concomitantly and affecting gene function during this period is bound to affect both pools of mRNAs, it has been challenging to study their expression dynamics separately. Results By employing SLAM-seq, a nascent mRNA labeling transcriptomic approach, in a developmental time series we observe that over half of the early zebrafish embryo transcriptome consists of maternal-zygotic genes, emphasizing their pivotal role in early embryogenesis. We provide an hourly resolution of de novo transcriptional activation events and follow nascent mRNA trajectories, finding that most de novo transcriptional events are stable throughout this period. Additionally, by blocking microRNA-430 function, a key post transcriptional regulator during zebrafish embryogenesis, we directly show that it destabilizes hundreds of de novo transcribed mRNAs from pure zygotic as well as maternal-zygotic genes. This unveils a novel miR-430 function during embryogenesis, fine-tuning zygotic gene expression. Conclusion These insights into zebrafish early embryo transcriptome dynamics emphasize the significance of post-transcriptional regulators in zygotic genome activation. The findings pave the way for future investigations into the coordinated interplay between transcriptional and post-transcriptional landscapes required for the establishment of animal cell identities and functions.

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Section: Discussionmentioning

confidence: 99%

miR-430 regulates zygotic mRNA during zebrafish embryogenesis

Baia Amaral,

Egidy,

Perera

et al. 2024

Genome Biol

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“…In many ways CasRx appears superior to RNAi. Numerous reports have also shown that CasRx can be successfully used in human cells ( Konermann et al, 2018 ), mice ( Zheng et al, 2022 ), and zebra fish ( Hernandez-Huertas et al, 2022 ), with no apparent cytotoxicity. However, one study found that CasRx caused in vivo toxicity to Drosophila melanogaster flies ( Buchman et al, 2020 ).…”

Section: Fidelity and Efficiency Of Clustered Regularly Interspersed ...mentioning

confidence: 99%

RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives

Goel

Ploski

2022

Front. Mol. Neurosci.

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Manipulating gene expression within and outside the nervous system is useful for interrogating gene function and developing therapeutic interventions for a variety of diseases. Several approaches exist which enable gene manipulation in preclinical models, and some of these have been approved to treat human diseases. For the last couple of decades, RNA interference (RNAi) has been a leading technique to knockdown (i.e., suppress) specific RNA expression. This has been partly due to the technology’s simplicity, which has promoted its adoption throughout biomedical science. However, accumulating evidence indicates that this technology can possess significant shortcomings. This review highlights the overwhelming evidence that RNAi can be prone to off-target effects and is capable of inducing cytotoxicity in some cases. With this in mind, we consider alternative CRISPR/Cas-based approaches, which may be safer and more reliable for gene knockdown. We also discuss the pros and cons of each approach.

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“…Over 70% of human genes have at least one zebrafish orthologue [ 36 ], with the Xenopus genome including orthologs of ~80% of human disease genes [ 37 ]. More recently, methods to increase the genome editing efficiency of the Clustered Regularly Interspaced Short Palindromic Repeat– (CRISPR–) Cas9 system in zebrafish [ 38 , 39 , 40 , 41 , 42 , 43 ] and Xenopus [ 44 , 45 , 46 , 47 , 48 ] have led to new human disease models.…”

Section: Aquatic Freshwater Vertebrate Animal Model Advantagesmentioning

confidence: 99%

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

Williams¹,

Mruk²

2022

IJMS

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Epilepsy is an international public health concern that greatly affects patients’ health and lifestyle. About 30% of patients do not respond to available therapies, making new research models important for further drug discovery. Aquatic vertebrates present a promising avenue for improved seizure drug screening and discovery. Zebrafish (Danio rerio) and African clawed frogs (Xenopus laevis and tropicalis) are increasing in popularity for seizure research due to their cost-effective housing and rearing, similar genome to humans, ease of genetic manipulation, and simplicity of drug dosing. These organisms have demonstrated utility in a variety of seizure-induction models including chemical and genetic methods. Past studies with these methods have produced promising data and generated questions for further applications of these models to promote discovery of drug-resistant seizure pathology and lead to effective treatments for these patients.

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Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos

Cited by 9 publications

References 9 publications

miR-430 regulates zygotic mRNA during zebrafish embryogenesis

miR-430 regulates zygotic mRNA during zebrafish embryogenesis

RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

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