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

Abstract

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 × 10⁶ P. 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.

Keywords: CRISPR/Cas9; Perkinsus marinus; heterologous expression system; oral adjuvant; oral vaccines; protozoan; transfection.

FIGURE 1

Plasmid amount and cell number optimization studies. (A) Fifty million parasites transfected with 5 μg (black bar), 10 μg (purple), 20 μg (blue), and 40 μg (orange) of pPmMOE[MOE1]:GFP, respectively. Bar graphs showing that the %GFP-positive cells (y-axis) were detected by flow cytometry at 24, 72, and 120 h post-transfection time points (x-axis). (B) Twenty-five million parasites transfected with 20 μg of pPmMOE[MOE1]:GFP 24 and 120 h post-transfections. (C) Twenty-five million parasites were transfected with 20 μg of pPmMOE[MOE1]:GFP, respectively. Bar graphs showing the %GFP-positive cells (y-axis) detected by flow cytometry at 24 (black bar), 72 (brown bar), 120 h (dark red bar), and 3 month (yellow bar) post-transfection time points (x-axis). (D) The scattered plot from FCM showing no GFP expression in untransfected controls and 98% GFP-positive cells in 25.0 × 10⁶ cells transfected with 20 μg of pPmMOE[MOE1]:GFP indicated in the green box.

FIGURE 2

Comparison of proprietary and non-proprietary transfection. Cells, 25 × 10⁶ cells transfected with 20 μg of pPmMOE[MOE1]:GFP plasmid using proprietary and non-proprietary protocols. (A) Flow cytometry scattered plot of untransfected (wild-type) cells, no GFP expression detected. (B) The scattered plot of flow cytometry, identifying GFP-positive cells in transfection performed using the proprietary Lonza method. (C) Scatterplot representation of GFP-positive cells in transfection performed using 3R buffer and BTX cuvette. (D) Scatterplot showing the GFP-positive cells when transfected with 3R buffer utilizing Lonza cuvette. (E) Bar graph showing the % of GFP-positive cells when transfected with the Lonza system (black bar), 3R buffer in combination with BTX cuvette (gray bar), 3R buffer using Lonza cuvette (dark gray), and Lonza buffer with BTX cuvette (white bar).

FIGURE 3

SpCas9-RNP and sgRNA-mediated GFP knock-in in P. marinus trophozoites. (A) Schematic representation of dDNA with 396 bp homology on the 5′ and 3′ of the GFP coding sequence. (B) Schematic representation of showing the guide RNA target sites on PmMOE1 coding sequence sgRNA-1 targets the top strand indicated by the arrow direction; sgRNA-2 targets the bottom strand indicated by the arrow direction. (C) Confocal microscopy panel showing successful GFP expression in cells transfected with sgRNA-1/SpCas9 and sgRNA-2/SpCas9, showing localization pattern similar to the PRA-393 MOE-GFP mutant strain. (D) The scattered plot from FCM showing no GFP expression in mock (dDNA+sgRNA alone) control and 0.2% GFP-positive cells knocked in using sgRNA-1, and 0.35% in case of sgRNA-2 indicated in the green box.

FIGURE 4

Sorting P. marinus GFP-positive cells for endogenous PmMOE1 C-terminus GFP tagging analysis. (A) Scattered plot showing 81% GFP-positive cells indicated with a green box in the experiment where cells transfected with sgRNA-1-Cas9. (B) Scattered plot showing 87% GFP-positive cells indicated with a green box in the experiment where cells transfected with sgRNA-1-Cas9. (C) The PCR intended to amplify the knock-in (expected sized 3,300 bp) using Fwd 1 and Rev 1 primers resulting in the amplification of the wildtype 2,600 bp amplicon (left panel). This PCR product was used as a template in the nested PCR (nPCR) to confirm the GFP knock-in using Fwd 2 and Rev 2 primers, which yielded the expected 748 bp amplicon (right panel), 1 kb Plus DNA Ladder (New England Biolabs, Ipswich, MA, United States). (D) Sequencing results of the nPCR product from the sgRNA-2 targeted GFP knock-in experiment.