skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus
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-DR4 0 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 p PmMOE[MOE1]:GFP and 25.0 × 10 6 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.  more » « less
Award ID(s):
1701480 1821065
PAR ID:
10250881
Author(s) / Creator(s):
; ; ; ; ; ;
Date Published:
Journal Name:
Frontiers in Bioengineering and Biotechnology
Volume:
9
ISSN:
2296-4185
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; (, Current Protocols)
    Abstract Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin‐relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off‐target effects are minimized by testing for the most efficient target site using a split‐GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split‐GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners 
    more » « less
  2. ; (, Trends in parasitology)
    null (Ed.)
    Perkinsus marinus is a facultative intracellular marine protozoan parasite responsible for the Dermo disease in Crassostrea virginica oysters. Associated with mass mortalities in the Gulf Coast and Chesapeake Bay (USA), it remains one of the main hurdles for oyster reef restoration and aquaculture. Oysters take up the parasite by filter-feeding; in the pallial cavity, it can be phagocytosed by the hemocytes via CvGal1, gaining access to the internal milieu. Inside the parasitophorous vacuole, the parasite resists oxidative stress and acquires nutrients. Propagation strategies include binary fission, budding, palintomy, and schizogony. Although the effect on humans upon consumption of raw infected oysters has not been studied, humanized HLA-DR4 mice fed with P. marinus do not develop noticeable pathology but elicit systemic immunity. Parasite culture in host-free media, and the use of genetic tools, make it a tractable genetic model and a heterologous expression and vaccine-delivery system. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; (, Advanced Functional Materials)
    Abstract CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. The RALA cell penetrating peptide is an amphiphilic peptide that self‐assembles into nanoparticles through electrostatic interactions with anionic molecules and delivers them across the cell membrane. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA effectively encapsulated and delivered CRISPR DNAs, RNAs, and ribonucleic proteins (RNPs) to primary mesenchymal stem cells (MSCs), outperforming commercially available reagents. We then used the RALA peptide for the knock‐in and knock‐out of reporter genes into primary MSCs and the transcriptional activation of therapeutically relevant genes. Finally, we demonstrate in vivo gene editing using RALA to knock‐out luciferase and GFP in a reporter mouse model. In summary, we establish RALA as a powerful tool for safer and more effective delivery of CRISPR machinery in multiple cargo formats for a wide range of ex vivo and in vivo gene editing strategies. 
    more » « less
  4. ; ; ; ; (, Molecular Therapy - Nucleic Acids)
    The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA sequencing to identify genes that may influence transfection efficiency in four different cell types (PC-3, Jurkat, HEK-293T, and primary T cells). The highest transfection efficiency was observed in HEK-293T cells, which upregulated only 142 genes with no known antiviral functions after transfection with lipofectamine. Lipofection upregulated 1,057 cytokine-stimulated genes (CSGs) in PC-3 cells, which exhibited a significantly lower transfection efficiency. However, when PC-3 cells were transfected in serum-containing media or electroporated, the observed transfection efficiencies were significantly higher while the expression levels of cytokines and CSGs decreased. In contrast, lipofection of Jurkat and primary T cells only upregulated a few genes, but several of the antiviral CSGs that were absent in HEK-293T cells and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain the relatively low Lipofection efficiencies observed with T cells (8%-21% GFP+). Indeed, overexpression of one CSG (IFI16) significantly decreased transfection efficiency in HEK-293T cells. 
    more » « less
  5. ; ; ; ; ; ; (, PLOS ONE)
    Schubert, Michael (Ed.)
    Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to testin vivoelectroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extendingin vivoelectroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email