CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Frontiers in Bioengineering and Biotechnology

José A. Fernández Robledo¹  Raghavendra Yadavalli¹  Adrienne N. Tracy²  Derek E. Hernández²  Asha V. Sidhu²  Kousuke Umeda³  Hannah A. Waugh⁴ 

[1] Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United States;Colby College, Waterville, ME, United States;National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan;Southern Maine Community College, South Portland, ME, United States;

关键词: Perkinsus marinus;    oral adjuvant;    heterologous expression system;    CRISPR/Cas9;    protozoan;    transfection;   

DOI  :  10.3389/fbioe.2021.623278

来源: DOAJ

【 摘 要 】

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

【 授权许可】

Unknown