CRISPR/Cas9 System-Mediated Gene Editing in the Fujian Oysters (Crassostrea angulate) by Electroporation

Abstract

The Fujian oyster (Crassostrea angulate) is an important marine bivalve mollusk with high economic value. Gene function research and gene editing techniques have broad application prospects in oyster. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for genome engineering in many species. CRISPR-mediated gene editing has also been used successfully in the Pacific oyster through direct delivery of the CRISPR/Cas9 components into oyster embryos by microinjection. However, the low throughput and operational difficulties associated with microinjection is one of the factors limiting the widespread application of CRISPR/Cas9 in oysters. In this study, we attempted to deliver the CRISPR/Cas9-system into the embryos of C. angulate by electroporation. An all-in-one CRISPR/Cas9 vector plasmid was used as CRISPR/Cas9 system in this study. Electroporation was carried out using both eggs and blastula larvae. A large number of larvae became malformed or die after electroporation. A single base substitution mutation was detected in the D-larvae developed from electroporated eggs. Our results demonstrate that the CRISPR/Cas9 system can be delivered into embryos of C. angulate for gene editing by electroporation, which provides a reference and will further contribute to the future application of electroporation in mollusks.

Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus

“…We employed electroporation for transfection, a highly effective transfection method for a variety of cells ( 55 ), considering the low proliferation rate, i.e., a less chance of the nuclear envelope breakdown, of scallop cells. In molluscs, electroporation has been used only in oyster embryos ( 13 , 16 ), and we report that molluscan primary cells were successfully electroporated with reporter genes.…”

“…S3 ). While a few studies carried out electroporation on bivalve embryos ( 13 , 14 , 16 ), there is no report of electroporation on molluscan primary cells. Thus, we first tested whether scallop primary cells could be transfected by electroporation using luciferase-encoding mRNA and optimized basal electroporation conditions ( Fig.…”

“…The efforts from the 1990s to transfect bivalve cells have not been very successful because of the low efficiency of transfection methods and the lack of strong promoters ( 8 ). In bivalve cells and embryos, the promoters tested so far are 5′ upstream regions of Drosophila hsp70 ( 9 , 10 ), Crassostrea gigas actβ ( 11 ), ef1α ( 12 – 14 ), the long terminal repeats of Molony murine leukemia virus ( 9 , 15 ), and the cytomegalovirus immediate early (CMV IE) promoter ( 14 , 16 , 17 ), but practically reliable promoters have yet to be discovered. Recently, applications of genome-editing techniques are advancing in bivalve research ( 13 , 18 ).…”

“…In bivalve cells and embryos, the promoters tested so far are 5′ upstream regions of Drosophila hsp70 ( 9 , 10 ), Crassostrea gigas actβ ( 11 ), ef1α ( 12 – 14 ), the long terminal repeats of Molony murine leukemia virus ( 9 , 15 ), and the cytomegalovirus immediate early (CMV IE) promoter ( 14 , 16 , 17 ), but practically reliable promoters have yet to be discovered. Recently, applications of genome-editing techniques are advancing in bivalve research ( 13 , 18 ). However, with the absence of efficient promoters, those studies are compelled to rely on direct delivery of genetic material by microinjection into small bivalve eggs ( 18 ) or electroporation with a conventional promoter ( 13 ).…”

“…Recently, applications of genome-editing techniques are advancing in bivalve research ( 13 , 18 ). However, with the absence of efficient promoters, those studies are compelled to rely on direct delivery of genetic material by microinjection into small bivalve eggs ( 18 ) or electroporation with a conventional promoter ( 13 ).…”