Data for SNSF Spark grant CRSK-3_190920: CRISPR-Cas9 gene editing to dissect the molecular mechanisms of rainbow trout antimicrobial peptides (AMPs)

Antimicrobial peptides (AMPs) are small amphipathic molecules (10-50 amino acids) that are produced by all life forms, from prokaryotes to humans, and are involved in the protection of the host against infections. Fish AMPs have rapidly captured the attention as novel drug candidates in aquaculture thanks to their broad antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi, parasites and viruses. In addition, their low propensity for the development of bacterial resistance makes AMPs a promising therapeutic alternative to the excessive and indiscriminate use of antibiotics in veterinary health. The molecular mechanism how AMPs exert their antimicrobial activity in fish cells is not fully understood. To overcome this lack of knowledge, we seek to identify the functional domains of AMPs in a rainbow trout (Oncorhynchus mykiss) gut cell line (RTgutGC). To achieve this aim, we propose to utilize an episomal CRISPR-Cas9 system to genetically screen for such functional AMP domains. The resulting gene-edited cell lines will be challenged with bacterial pathogens, such as Streptococcus dysgalactiae and Yersinia ruckeri and the ability of these gene-edited cell lines to overcome the bacterial burden will be assessed. Moreover, a time-course transcriptome (RNA-seq) and proteome (mass spectrometry) analysis of RTgutGC cells that express AMPs upon exposure to synthetic bacterial molecules will be conducted. Taking together, these findings will give us the opportunity to shed light on the molecular mechanisms that drive the therapeutic effect of AMPs in fish cells and to gain a global view on the molecular pathways that are affected when AMPs are expressed.