ZEBRAFISH AND CRISPR/CAS: A MODEL TO ELUCIDATE GENETIC EFFECTS ON THE MICROBIOTA

Introduction

 With the global population growing, the need for high-quality sustainably produced protein sources for human consumption is bound to increase. It is therefore important to develop novel, innovative and sustainable solutions to produce food and promote growth. Aquaculture will play a prominent role in that aspect. While probiotics for use in aquaculture have emerged as one possible solution for modulating the microbiota to promote growth, their effects remain inconclusive and uncertain. This may lie in the fact that microbiota research has largely been focused on the effects of microbiota on its host, rather than the potential effects host genetics have on its microbiota.

 Elucidating potential host genetic effects on its microbiota has obvious biotechnological applications in the aquaculture industry. For example, designed feed to suit and promote growth of a particular genotype or genetic engineering of the host to promote a healthy microbiome. The field of applied hologenomics using zebrafish (Danio rerio ) as a model organism provides a promising framework to study such effects.

Here, we applied a 16S metabarcoding approach in combination with a CRISPR/Cas mediated knockout of the gene coding for tyrosinase (tyr), the rate-limiting enzyme in melanogenesis, to provide a proof-of-principle that zebrafish can be used to study the effects of host genetics on its microbiota.

Methods

 We generated a F0 generation of zebrafish by using CRISPR/Cas-9 to knock-out the tyr gene in the zebrafish genome. Phenotypes were classified into two groups based on pigmentation levels: albino and mosaic. The phenotypic groups were housed in separate tanks. The microbial community of faeces samples collected from the tanks containing the different pigmentation phenotypes was assessed with the bacterial 16S rRNA gene. The microbial communities of the two mutant pigmentation phenotype groups were compared to the microbial community of a third group with a wild type pigmentation pattern (figure 1).

Results

 The knock-out of a single gene, tyr , using CRISPR/Cas-9, resulted in changes in pigmentation and in the zebrafish intestinal microbiota. Both alpha-diversity and beta-diversity differed significantly among the pigmentation phenotypes. The differences were mainly driven by the relative abundance of two genera: Cetobacterium and Aeromonas . Along with these genera, Pseduomonas and Vibrio were detected as differentially abundant among the pigmentation phenotypes. These genera may be implicated in immune- and metabolic-related mechanisms.

Conclusions

The results suggest an effect of tyrosinase function on the intestinal microbiota community of zebrafish. Overall, the study shows that the zebrafish presents a satisfactory model to study host-genomic effects on the microbiota and that further studies can shed even more light on such effects. Future studies should include a more multi-omic approach to gain more functional insights from the interactions between the host genetics and its microbiota. Moreover, such studies could prove highly valuable to the aquaculture industry. This could be in the form of probiotics tailored to the genotypes of broodstock fish, or perhaps the selection of genotypes that code for a healthy microbiota community.