Development of an RNA anti-parasite based on CRISPR-Cas9 against Chilodonella piscicola

Abstract

Chilodonella piscicola is a gill and body surface parasite of freshwater fish, affecting the growth of juvenile and adult freshwater fishes. To develop an effective method to manage C.piscicola in aquaculture, we used CRISPR/Cas9 technology to design and validate small guide RNA targeting DNA barcoding ITS-1 and 18S rDNA sequences of C.piscicola to develop a species-specific pesticide. In this study, effective sgRNA targets were found in ITS-1 and 18S rDNA sequences of C.piscicola, respectively. In the soaking experiment, we found that the mixing of Cas9 mRNA and sgRNAs could significantly reduce the survival rate of C.piscicola. The Real-time quantitative PCR results showed that both Cas9 works with the single sgRNA or the mixture of the two sgRNA could destroy the parasite DNA at a specific region. DNA sequencing also verified the effectiveness of single sgRNA or two sgRNAs mixed with cas9 induced mutation on the target fragment of the parasite DNA in the experimental group. The results of this study once again verified the effectiveness of genetic barcoding in the application of anti-parasitic and provided a new idea for anti-parasitic methods.