Sterilization of fish through adaptable gRNAs targeting dnd1 using CRISPR-Cas13d system

Abstract

Sterilizing fish is a valuable technique not only for preventing genetic contamination from escaped aquaculture fish but also for enhancing fish meat quality. The clustered regularly interspaced short palindromic repeats-CRISPR-associated 13d (CRISPR-Cas13d) system is capable of RNA-targeted gene knockdown with reduced off-target effects. Unlike morpholino knockdown, which is limited to specific target sites, the CRISPR-Cas13d system utilizes the entire mRNA as a target site for gRNA. This enables the design of adaptable gRNAs by targeting the conserved sequences across fish species, leading to promising sterilization for a wide variety of aquaculture fish. In this study, we demonstrated that the effectiveness of this system in fish sterilization by the knockdown of dead end1 (dnd1) and nanos3, which are crucial for germ cell development. Initially, we demonstrated that gRNAs designed using the cas13design tool, which was developed based on studies of cultured mammalian cells, effectively induced germ cell-deficient medaka with low-cytotoxicity. Additionally, we identified the nucleotide positions of the gRNA (seed sequence) that are crucial for knockdown in teleost fish. These data will be valuable for designing adaptable gRNA in teleost fish and targeting multiple paralogous genes. To develop an adaptable sterilization technique using the CRISPR-Cas13d system, we aligned 81 dnd1 sequences from 70 fish species and identified a highly conserved region of the gRNA targets. Despite not finding a perfectly matched target region in the dnd1 alignment, we designed 15 adaptable gRNAs that potentially targeting dnd1 in 55 out of 70 fish species, leveraging sequences from representative aquaculture and aquarium fish (salmon, tilapia, puffer fish, amberjack, tuna, guppy and carp) and experimental model fish (medaka, zebrafish, and stickleback. To confirm whether adaptable gRNAs had a knockdown effect, we initially injected the gRNAs into medaka embryos. Adaptable gRNAs with a single-nucleotide mismatch against medaka dnd1 resulted in the complete loss of germ cells with little cytotoxicity, suggesting that mismatches are unlikely to induce off-target effects crucial for embryonic development. Additionally, adaptable gRNAs designed based on the salmon dnd1 sequence, which was 100% identical in the Oncorhynchus, Salvelinus, and Salmo groups, effectively generated germ cell-deficient rainbow trout. These results indicate that the knockdown of dnd1 using the CRISPR-Cas13d system is effective for sterilizing both aquaculture and experimental model fish species. Moreover, the adaptable gRNAs designed in this study can potentially sterilize a diverse range of fish species.