Multi-locus gene editing effectively knocked out cyp19a1a and foxl2 in Monopterus albus, a hermaphroditic fish

Homozygous mutant populations are typically obtained when studying gene function using gene editing. However, because of asynchronous testicular and ovarian development, it is difficult to obtain homozygous mutant offspring of hermaphrodite fish by mating males and females. The rice field eel (Monopterus albus) was the first hermaphrodite teleost to be identified. Natural sex change characteristics render M. albus a good model for studying vertebrate sex differentiation. Rice field eels are also an economically important aquaculture species in East and Southeast Asia. In this study, we established a highly efficient multi-locus gene editing method for rice field eels. We selected cyp19a1a and foxl2 for editing and targeted seven and 11 sites in the coding regions of these genes, respectively. Fertilized eggs were co-injected with the Cas9 protein and the guide RNAs of each gene. The efficiency of gene editing-induced mutations in cyp19a1a and foxl2 was approximately 100% in the microinjected embryos and adult fish tissues. In cyp19a1a mutants, serum E2 levels were decreased, and ovarian development was blocked. Only germ stem cells or oogonia were present in the gonads of the 12-month-old cyp19a1a mutant fish. These results suggest that cyp19a1a plays a key role in regulating primary gonadal development in the ovaries of rice field eels. In foxl2 mutants, cyp19a1a transcription and serum E2 levels were minimally decreased, whereas the transcription of foxl2-l, foxl3, and dmrt1 was increased. Nevertheless, the gonads of foxl2 mutant fish differentiated into ovaries, thereby indicating that foxl2 does not directly affect ovarian development in rice field eels. The multi-locus gene editing method established in this study provides an effective approach for studying gene function study and improving the genetic traits in rice field eels.