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Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing

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Malaria Control and Elimination

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2013))

Abstract

Malaria continues to be a global health burden, threatening over 40% of the world’s population. Drug resistance in Plasmodium falciparum, the etiological agent of the majority of human malaria cases, is compromising elimination efforts. New approaches to treating drug-resistant malaria benefit from defining resistance liabilities of known antimalarial agents and compounds in development and defining genetic changes that mediate loss of parasite susceptibility. Here, we present protocols for in vitro selection of drug-resistant parasites and for site-directed gene editing of candidate resistance mediators to test for causality.

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References

  1. World Health Organization (2018) World malaria report 2018. https://www.who.int/malaria/publications/world-malaria-report-2018/report/en

  2. Dondorp AM, Nosten F, Yi P et al (2009) Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361:455–467

    Article  CAS  Google Scholar 

  3. Ashley EA, Dhorda M, Fairhurst RM et al (2014) Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 371:411–423

    Article  Google Scholar 

  4. Takala-Harrison S, Jacob CG, Arze C et al (2015) Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis 211:670–679

    Article  CAS  Google Scholar 

  5. Tun KM, Imwong M, Lwin KM et al (2015) Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis 15:415–421

    Article  CAS  Google Scholar 

  6. Sonoiki E, Ng CL, Lee MC et al (2017) A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue. Nat Commun 8:14574

    Article  Google Scholar 

  7. Vanaerschot M, Lucantoni L, Tao L et al (2017) Hexahydroquinolines are antimalarial candidates with potent blood stage and transmission-blocking activity. Nat Microbiol 2:1403–1414

    Article  CAS  Google Scholar 

  8. Cowell AN, Istvan ES, Lukens AK et al (2018) Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics. Science 359:191–199

    Article  CAS  Google Scholar 

  9. Sidhu AB, Verdier-Pinard D, Fidock DA (2002) Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298:210–213

    Article  CAS  Google Scholar 

  10. de Koning-Ward TF, Gilson PR, Crabb BS (2015) Advances in molecular genetic systems in malaria. Nat Rev Microbiol 13:373–387

    Article  Google Scholar 

  11. Straimer J, Lee MC, Lee AH et al (2012) Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nat Methods 9:993–998

    Article  CAS  Google Scholar 

  12. Wagner JC, Platt RJ, Goldfless SJ et al (2014) Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum. Nat Methods 11:915–918

    Article  CAS  Google Scholar 

  13. Ghorbal M, Gorman M, Macpherson CR et al (2014) Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 32:819–821

    Article  CAS  Google Scholar 

  14. Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  Google Scholar 

  15. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

    Article  CAS  Google Scholar 

  16. Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

    Article  CAS  Google Scholar 

  17. Lee AH, Symington LS, Fidock DA (2014) DNA repair mechanisms and their biological roles in the malaria parasite Plasmodium falciparum. Microbiol Mol Biol Rev 78:469–486

    Article  CAS  Google Scholar 

  18. Kirkman LA, Lawrence EA, Deitsch KW (2014) Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity. Nucleic Acids Res 42:370–379

    Article  CAS  Google Scholar 

  19. Zhang C, Xiao B, Jiang Y et al (2014) Efficient editing of malaria parasite genome using the CRISPR/Cas9 system. MBio 5:e01414–e01414

    PubMed  PubMed Central  Google Scholar 

  20. Ganesan SM, Morrisey JM, Ke H et al (2011) Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection. Mol Biochem Parasitol 177:29–34

    Article  CAS  Google Scholar 

  21. Fidock DA, Wellems TE (1997) Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. Proc Natl Acad Sci U S A 94:10931–10936

    Article  CAS  Google Scholar 

  22. Duraisingh MT, Triglia T, Cowman AF (2002) Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. Int J Parasitol 32:81–89

    Article  CAS  Google Scholar 

  23. LaMonte G, Lim MY, Wree M et al (2016) Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) confer multidrug resistance. MBio 7:e00696–e00616

    Article  CAS  Google Scholar 

  24. Lim MY, LaMonte G, Lee MC et al (2016) UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes. Nat Microbiol 1:16166

    Article  CAS  Google Scholar 

  25. Ng CL, Siciliano G, Lee MC et al (2016) CRISPR-Cas9-modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine-containing compounds but potentiates artemisinin-based combination therapy partner drugs. Mol Microbiol 101:381–393

    Article  CAS  Google Scholar 

  26. Crawford ED, Quan J, Horst JA et al (2017) Plasmid-free CRISPR/Cas9 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733. PLoS One 12:e0178163

    Article  Google Scholar 

  27. Bansal A, Ojo KK, Mu J et al (2016) Reduced activity of mutant calcium-dependent protein kinase 1 is compensated in Plasmodium falciparum through the action of protein kinase G. MBio 7:e02011–e02016

    Article  CAS  Google Scholar 

  28. Mogollon CM, van Pul FJ, Imai T et al (2016) Rapid generation of marker-free P. falciparum fluorescent reporter lines using modified CRISPR/Cas9 constructs and selection protocol. PLoS One 11:e0168362

    Article  Google Scholar 

  29. Kuang D, Qiao J, Li Z et al (2017) Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9. Parasit Vectors 10:595

    Article  Google Scholar 

  30. Miliu A, Lebrun M, Braun-Breton C et al (2017) Shelph2, a bacterial-like phosphatase of the malaria parasite Plasmodium falciparum, is dispensable during asexual blood stage. PLoS One 12:e0187073

    Article  Google Scholar 

  31. Nacer A, Claes A, Roberts A et al (2015) Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes. Cell Microbiol 17:1205–1216

    Article  CAS  Google Scholar 

  32. Bansal A, Molina-Cruz A, Brzostowski J et al (2018) PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection. Proc Natl Acad Sci U S A 115:774–779

    Article  CAS  Google Scholar 

  33. Bryant JM, Regnault C, Scheidig-Benatar C et al (2017) CRISPR/Cas9 genome editing reveals that the intron is not essential for var2csa gene activation or silencing in Plasmodium falciparum. MBio 8:e00729–e00717

    Article  Google Scholar 

  34. Wezena CA, Alisch R, Golzmann A et al (2017) The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development. Microb Cell 5:32–41

    Article  Google Scholar 

  35. Bansal A, Molina-Cruz A, Brzostowski J et al (2017) Plasmodium falciparum calcium-dependent protein kinase 2 is critical for male gametocyte exflagellation but not essential for asexual proliferation. MBio 8:e01656-17

    Article  Google Scholar 

  36. Cobb DW, Florentin A, Fierro MA et al (2017) The exported chaperone PfHsp70x is dispensable for the Plasmodium falciparum intraerythrocytic life cycle. mSphere 2:e00363–e00317

    Article  Google Scholar 

  37. Knuepfer E, Napiorkowska M, van Ooij C et al (2017) Generating conditional gene knockouts in Plasmodium - a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9. Sci Rep 7:3881

    Article  Google Scholar 

  38. Walczak M, Ganesan SM, Niles JC et al (2018) ATG8 is essential specifically for an autophagy-independent function in apicoplast biogenesis in blood-stage malaria parasites. MBio 9:e02021–e02017

    Article  CAS  Google Scholar 

  39. Nasamu AS, Glushakova S, Russo I et al (2017) Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion. Science 358:518–522

    Article  CAS  Google Scholar 

  40. Sidik SM, Huet D, Ganesan SM et al (2016) A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166:1423–1435

    Article  CAS  Google Scholar 

  41. Zhang C, Li Z, Cui H et al (2017) Systematic CRISPR-Cas9-mediated modifications of Plasmodium yoelii ApiAP2 genes reveal functional insights into parasite development. MBio 8:e01986–e01917

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhang C, Gao H, Yang Z et al (2017) CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii. Mol Biochem Parasitol 212:1–8

    Article  CAS  Google Scholar 

  43. Doyon Y, Vo TD, Mendel MC et al (2011) Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8:74–79

    Article  CAS  Google Scholar 

  44. Miller JC, Holmes MC, Wang J et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785

    Article  CAS  Google Scholar 

  45. McNamara CW, Lee MC, Lim CS et al (2013) Targeting Plasmodium PI(4)K to eliminate malaria. Nature 504:248–253

    Article  CAS  Google Scholar 

  46. Straimer J, Gnadig NF, Witkowski B et al (2015) K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science 347:428–431

    Article  CAS  Google Scholar 

  47. Gabryszewski SJ, Modchang C, Musset L et al (2016) Combinatorial genetic modeling of pfcrt-mediated drug resistance evolution in Plasmodium falciparum. Mol Biol Evol 33:1554–1570

    Article  CAS  Google Scholar 

  48. Fidock DA, Nomura T, Wellems TE (1998) Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase. Mol Pharmacol 54:1140–1147

    Article  CAS  Google Scholar 

  49. Ekland EH, Schneider J, Fidock DA (2011) Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. FASEB J 25:3583–3593

    Article  CAS  Google Scholar 

  50. MacPherson CR, Scherf A (2015) Flexible guide-RNA design for CRISPR applications using Protospacer Workbench. Nat Biotechnol 33:805–806

    Article  CAS  Google Scholar 

  51. Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418–420

    Article  CAS  Google Scholar 

  52. Ran FA, Cong L, Yan WX et al (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Marcus Lee for his development of several T7- and U6-based editing systems that are discussed herein.

Author information

Authors and Affiliations

  1. Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA

    Caroline L. Ng

  2. Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, USA

    David A. Fidock

  3. Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA

    David A. Fidock

Authors
  1. Caroline L. Ng
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  2. David A. Fidock
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Corresponding author

Correspondence to David A. Fidock .

Editor information

Editors and Affiliations

  1. INSERM 1016 Institut Cochin, Service de Parasitologie-Mycologie, Université Paris Descartes, Hôpital Cochin, Paris, France

    Frédéric Ariey

  2. Faculté de Médecine, AP-HP, Sorbonne Université, Groupe Hospitalier Pitié-Salpêtrière, Paris, France

    Frédérick Gay

  3. Malaria Infection and Immunity Unit, Institut Pasteur, Paris, France

    Robert Ménard

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Ng, C.L., Fidock, D.A. (2019). Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing. In: Ariey, F., Gay, F., Ménard, R. (eds) Malaria Control and Elimination. Methods in Molecular Biology, vol 2013. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9550-9_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9550-9_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9549-3

  • Online ISBN: 978-1-4939-9550-9

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