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Genetic Manipulation of Cryptosporidium parvum with CRISPR/Cas9

Adam Sateriale1, Mattie Pawlowic2, Sumiti Vinayak3

  • 1Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|August 28, 2019
PubMed

Insights

Genetic manipulation of Cryptosporidium parvum is now reliable using CRISPR/Cas9 and mouse models. Simplified protocols streamline the process for creating transgenic parasites.

Area of Science:

  • Parasitology
  • Molecular Biology
  • Genetics

Background:

  • Cryptosporidium parvum is an opportunistic pathogen causing significant gastrointestinal illness.
  • Genetic manipulation of Cryptosporidium parvum has historically been challenging.
  • CRISPR/Cas9 technology offers a powerful tool for genetic modification.

Purpose of the Study:

  • To provide a detailed guide for genetically manipulating Cryptosporidium parvum.
  • To outline a simplified and reliable protocol for generating transgenic strains.
  • To facilitate research into Cryptosporidium parvum biology and pathogenesis.

Main Methods:

  • Utilizing CRISPR/Cas9-driven homologous repair for genetic modification.
  • Employing in vivo propagation within immunodeficient mice for parasite maintenance.
  • Describing key procedures including excystation, transfection, infection, collection, and purification.

Main Results:

  • Demonstrated reliable genetic manipulation of Cryptosporidium parvum.
  • Reported significant simplification of the initial genetic modification protocol.
  • Established a comprehensive workflow for generating and purifying transgenic parasites.

Conclusions:

  • CRISPR/Cas9 homologous repair is an effective method for Cryptosporidium parvum genetic modification.
  • The simplified protocol enhances the accessibility and efficiency of creating transgenic strains.
  • This methodology will advance research on Cryptosporidium parvum, aiding in the development of control strategies.
Keywords:
CRISPRCas9GeneticsTransfectionTransgenics

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