Genetic Manipulation of Cryptosporidium parvum with CRISPR/Cas9

  • Adam Sateriale
  • Mattie Pawlowic
  • Sumiti Vinayak
  • Carrie Brooks
  • Boris StriepenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2052)


Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9-driven homologous repair coupled to in vivo propagation within immunodeficient mice. Recent modifications have simplified the initial protocol significantly. This chapter will guide through procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum.


Transfection Transgenics CRISPR Cas9 Genetics 



Our work on Cryptosporidium transgenesis was supported by grants from the National Institutes of Health, the Wellcome Trust, and the Bill & Melinda Gates Foundation to B.S. MCP was supported by NIH T32AI060546 training grant, and AS received F32AI116053 and K99AI37442 career fellowships from the National Institutes of Health.


  1. 1.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. Scholar
  2. 2.
    van Gent DC, Hoeijmakers JH, Kanaar R (2001) Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet 2(3):196–206. Scholar
  3. 3.
    Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304(5669):441–445. Scholar
  4. 4.
    Vinayak S, Pawlowic MC, Sateriale A, Brooks CF, Studstill CJ, Bar-Peled Y, Cipriano MJ, Striepen B (2015) Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523(7561):477–480. Scholar
  5. 5.
    Pawlowic MC, Vinayak S, Sateriale A, Brooks CF, Striepen B (2017) Generating and maintaining transgenic Cryptosporidium parvum parasites. Curr Protoc Microbiol 46:20B 22 21–20B 22 32. Scholar
  6. 6.
    Robertson LJ, Campbell AT, Smith HV (1993) In vitro excystation of Cryptosporidium parvum. Parasitology 106:13–19CrossRefGoogle Scholar
  7. 7.
    Jothikumar N, da Cilva AJ, Moura I, Qvarnstrom Y, Hill VR (2008) Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays. J Med Microbiol 57(Pt 9):1099–1105. Scholar
  8. 8.
    Arrowood MJ, Donaldson K (1996) Improved purification methods for calf-derived Cryptosporidium parvum oocysts using discontinuous sucrose and cesium chloride gradients. J Eukaryot Microbiol 43(5):89SCrossRefGoogle Scholar
  9. 9.
    Peng D, Tarleton R (2015) EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens. Microb Genom 1(4):e000033. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Adam Sateriale
    • 1
  • Mattie Pawlowic
    • 2
  • Sumiti Vinayak
    • 3
  • Carrie Brooks
    • 4
  • Boris Striepen
    • 1
    Email author
  1. 1.Department of Pathobiology, School of Veterinary MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Wellcome Trust Centre for Anti-Infectives ResearchUniversity of DundeeDundeeUK
  3. 3.Department of Pathobiology, College of Veterinary MedicineUniversity of Illinois Urbana-ChampaignUrbanaUSA
  4. 4.Center for Tropical and Emerging and Global DiseasesUniversity of GeorgiaAthensUSA

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