Establishment of CRISPR-Cas-based antiparasitic agents for the swimming crab parasite Mesanophrys sp.

Mesanophrys sp. represents a parasitic ciliate, significantly jeopardizing the survival of farmed swimming crabs (Portunus trituberculatus). However, certain drawbacks exist in traditional broad-spectrum antiparasitic drugs, necessitating the development of innovative antiparasitic methods. Here we report the development of a series of CRISPR-Cas-based antiparasitic engineering bacteria, termed AEB-Cas(s), capable of sequence-specific killing of Mesanophrys sp.. The AEB-Cas(s) are generated by introducing well-designed prokaryotic plasmid vectors into chassis bacteria to express CRISPR effector protein and guide RNA targeting the 18S rDNA, 28S rDNA, and DHC7 genes of ciliates. By virtue of the bacteria-feeding behavior of ciliates, a feeding-delivery (FD) strategy is established for the targeted delivery of exogenous proteins and RNAs into Mesanophrys sp.. Based on the FD strategy, AEB-Cas9 and AEB-Cas13 antiparasitic agents are feeding to the ciliates, and their antiparasitic effect on Mesanophrys sp. are evaluated, respectively. Our results demonstrate that AEB-Cas(s) exhibit significant differences in the inhibitory effect when targeting different ciliates' genes. Specifically, AEB-Cas9 and AEB-Cas13 exhibit 39.0% and 52.2% inhibition rates targeting the 18S rDNA gene, respectively. In comparison, the inhibition rates are 55.2% and 73.6% targeting the 28S rDNA gene, respectively. Notably, both exhibit the highest lethality targeting the DHC7 gene, with inhibition rates of 56.5% in AEB-Cas9 and 89.4% in AEB-Cas13, respectively. Our findings highlight the potential of AEB-Cas(s) as effective antiparasitic agents against Mesanophrys sp., and provide a promising approach for preventing and treating other aquatic ciliate diseases.