Construction of a cytidine base editor based on Exopalaemon carinicaudacytidine deaminase and its application in crustacean genome editing

Economic crustaceans play important roles in aquaculture and create great benefits for the world agricultural economy. Studies have found that there are many single nucleotide polymorphism (SNP) sites in the crustacean genomes, which closely related to a variety of life activities like growth, development, reproduction, and so on. However, the in vivofunctional study of SNPs still lacks appropriate technique in crustaceans. The cytosine base editor (CBE) based on cytidine deaminase (CDA) can convert the C•G base pair to T•A base pair, which provides a new idea for the study of function of crustacean SNPs. In this study, the CDA gene from Exopalaemon carinicauda(EcCDA) was cloned and fused to the N-terminus of Cas9 nickase (nCas9(D10A)) skeleton to construct a cytidine base editor (pEcCDA-nCas9(D10A)) suitable for crustacean point editing. Neocaridina denticulata sinensisMIH (NdMIH) gene was selected as the target gene. The in vitrotranscribed EcCDA-nCas9(D10A) mRNA and NdMIH-gRNA were co-injected into the 1-cell stage embryos of N. denticulata sinensisfor gene editing. Detection results showed that the constructed pEcCDA-nCas9(D10A) successfully realized the conversion of C•G to T•A in NdMIHwith the editing efficiency of approximately 11.21%. Our works construct a well-established crustacean base editing platform.