In vitro bioassays for pro- and retrospective environmental toxicity testing

De novo establishment of in vitro fish bioassays via CRISPR/Cas-mediated reporter gene knock-ins for pro- and retrospective environmental toxicity testing. Project leader: Sebastian Lungu Mitea

Pro- and retrospective regulatory toxicity testing accounts for approximately 25% of animals used in research. Besides rodents, fish are the second most applied model. Efforts have been undertaken within human toxicology to transform the discipline – from a lethality-driven to a mechanistically-driven one. Many new approach methods, including in vitro and in silico test systems, were developed. However, within ecotoxicology, this process is in its infancy. Many mammalian and bacterial bioassay were adapted to compensate for the latter but lack ecological relevance and are technically problematic. Recent studies disclosed that cellular reporters derived via classic gene-engineering and transgenesis technologies might be subject to artefacts.

Contrarily, the CRISPR/Cas tools avoid such pitfalls, given their high resolution and accuracy. Here, we use this technology to design novel reporter gene assays to assess primary toxicity pathways in zebrafish cell lines. The idea is to drive reporter gene expression by endogenous genomic regulatory elements within their original loci. Thereby, an actual physiological response can be recorded without drastically alternating the cellular system under investigation.

Further, the approach is supplemented by computational modeling, such as mass balance equations and in vitro to in vivo extrapolations. Modelling is utilized to compute actual bioavailable effect concentrations in vivo from nominal exposure concentrations in vitro. Thereby, regulatory acceptance could be fulfilled, leading to the partial or total replacement of acute and chronic regulatory in vivo testing in an ecotoxicological context. We regard such in vitro assays as a technological improvement within the field of ecotoxicology, with the potential to drastically decrease the use of fish in regulatory toxicity testing.

This project has received 2 million SEK from the U-share project at the disciplinary domain of medicine and pharmacy, UU ( and will be conducted in a collaboration with colleagues from SLU (molecular toxicology) and KI (genome engineering lab) (

Last modified: 2023-04-12