Author: Del Prado, Joaquin Abugattas-Nuñez ; Málaga-Trillo, Edward ; Moreno-Mateos, Miguel A ; Spaink, Herman P ; der Kolk, Kees-Jan van ; Sonneville, Jan de ; Ding, Yi

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.

Cold Spring Harbor Perspectives in Medicine

Zebrafish Models for Drug Discovery and Therapeutic Validation against Non-Tuberculous Mycobacteria

Article

Author: Hamela, Claire ; Ding, Yi ; Johansen, Matt D ; Kremer, Laurent

The incidence of non-tuberculous mycobacteria (NTM) is increasing globally, often surpassing the incidence of new tuberculosis (TB) cases in developed countries. Most NTM are environmental organisms; however, there are a number of opportunistic and pathogenic species that can cause severe infections in animals and humans. Many NTM are intrinsically resistant to anti-TB therapies and are incredibly difficult to treat, resulting in poor treatment outcomes for these patients. Recent advances in preclinical animal models such as the zebrafish models have led to the discovery of highly active antimicrobial and host-directed therapies (HDTs) targeting NTM infections that can be applied to treat human infections. Here, we summarize recent progress and technological advancements in the discovery and development of antimicrobial drugs and HDTs that have been applied to NTM zebrafish infection models. We highlight the future directions for this increasingly applicable animal model for the discovery of next-generation therapies to treat NTM diseases.

Journal of Visualized ExperimentsQ4 · CROSS-FIELD

Establishment and Optimization of a High Throughput Setup to Study <em>Staphylococcus epidermidis</em> and <em>Mycobacterium marinum</em> Infection as a Model for Drug Discovery

Q4 · CROSS-FIELD

Article

Author: Veneman, Wouter J ; de Sonneville, Jan ; Ordas, Anita ; Marín-Juez, Rubén ; Meijer, Annemarie H ; Spaink, Herman P ; Jong-Raadsen, Susanne

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

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