Identification of active shrimp U6 promoters for efficient CRISPR/Cas9 gene editing in penaeid shrimps based on a VP28-pseudotyped baculovirus expression system

Abstract

The activity of species-specific U6 promoter is crucial for efficient expression of sgRNA in target cells when CRISPR/Cas9 is delivered in plasmid, thus the use of CRISPR/Cas9 technology was markedly limited in shrimps due to the lack of endogenous U6 promoter. To overcome this, in this study four candidate U6 promoters (LvU6 A-D) had been identified and cloned from the genome of penaeid shrimp (Litopenaeus vannamei), and then their activities to drive the expression of shRNA in insect Sf9 cells and shrimp primary hemolymph cells were successively analyzed using insect baculovirus expression system. It was found that all the isolated four LvU6 promoters contained all the characteristic elements (DSE, PSE and TATA box) of RNA polymerase III U6 promoter. Both a dual plasmids delivery system (Bacmid-PH-LvU6-shEGFP-P2-gBFP and Bacmid-PH-EGFP) and an all-in-one plasmid delivery system (Bacmid-PH-EGFP-LvU6-shEGFP-P2-gBFP) were constructed for RNAi analysis of reporter gene EGFP which are not pseudo-typed for the infection of Sf9 cells but pseudo-typed for the infection of shrimp cells by co-transfection with pFastBac1-VP28 (an envelope protein of shrimp virus). The obtained results showed that only LvU6 A, B and D could successfully drive the expression of shEGFP in both of the tested cells, and the order of interference efficiency was LvU6 B > LvU6 D > LvU6 A. Furthermore, the RNAi efficiency of LvU6 B was up to 27% (P < 0.05) when the dual plasmids were co-transfected at a mass ratio of 1: 0.1 in Sf9 cells, which was higher than that of the all-in-one plasmid (15%, P < 0.05). In shrimp cells, no noticeable fluorescence could be observed when dual plasmids delivery system was used; in contrast, the efficiency could reach 19% when all-in-one delivery system was used (P < 0.05). However, when LvU6 B was used in the shrimp CRISPR/Cas9 gene editing system with a co-expressed VP28 gene in the baculovirus vector, there were no mutations detected in the target gene (Bax) of both in vitro cultured shrimp cells and adult shrimps. Surprisingly, after optimizing the system by using a truncated LvU6 B promoter and introducing double sgRNAs, the insertion, deletion or replacement of target sites occurred in both shrimp cells and adult tissues (spermatophore, Oka and intestine), and the mutation rates increased up to 22% and 46%, respectively. This work has provided one active shrimp U6 promoter for efficient gene editing in shrimps and shrimp cells.