Genome Editing in the Human Malaria Parasite Plasmodium Falciparum Using the CRISPR-Cas9 System

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2014 Jun 2

PMID 24880488

Citations 358

Authors

Mehdi Ghorbal

Molly Gorman

Cameron Ross Macpherson

Rafael Miyazawa Martins

Artur Scherf

Jose-Juan Lopez-Rubio

Affiliations

Soon will be listed here.

Abstract

Genome manipulation in the malaria parasite Plasmodium falciparum remains largely intractable and improved genomic tools are needed to further understand pathogenesis and drug resistance. We demonstrated the CRISPR-Cas9 system for use in P. falciparum by disrupting chromosomal loci and generating marker-free, single-nucleotide substitutions with high efficiency. Additionally, an artemisinin-resistant strain was generated by introducing a previously implicated polymorphism, thus illustrating the value of efficient genome editing in malaria research.

Citing Articles

Maintenance of pyrophosphate homeostasis in multiple subcellular compartments is essential in .

Nwankwo I, Ke H bioRxiv. 2025; .

PMID: 40027813 PMC: 11870574. DOI: 10.1101/2025.02.20.639246.

Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei.

Das S, Unhale T, Marinach C, Valeriano Alegria B, Roux C, Madry H Sci Rep. 2025; 15(1):2949.

PMID: 39849074 PMC: 11758014. DOI: 10.1038/s41598-025-87114-4.

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei.

Jonsdottir T, Paoletta M, Ishizaki T, Hernandez S, Ivanova M, Herrera Curbelo A Nucleic Acids Res. 2025; 53(2.

PMID: 39844455 PMC: 11754126. DOI: 10.1093/nar/gkaf005.

transcription factor AP2-06B is mutated at high frequency in Southeast Asia but does not associate with drug resistance.

Shi Q, Wang C, Yang W, Ma X, Tang J, Zhang J Front Cell Infect Microbiol. 2025; 14():1521152.

PMID: 39835275 PMC: 11744005. DOI: 10.3389/fcimb.2024.1521152.

Identification of an artemisinin-resistant kelch13 R515K mutant parasite in Senegal.

Sene S, Pouye M, Martins R, Diallo F, Mangou K, Bei A Front Parasitol. 2025; 2():1076759.

PMID: 39816817 PMC: 11731788. DOI: 10.3389/fpara.2023.1076759.

References

Ganesan S, Morrisey J, Ke H, Painter H, Laroiya K, Phillips M . Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection. Mol Biochem Parasitol. 2011; 177(1):29-34. PMC: 3057331. DOI: 10.1016/j.molbiopara.2011.01.004. View

Pfander C, Anar B, Schwach F, Otto T, Brochet M, Volkmann K . A scalable pipeline for highly effective genetic modification of a malaria parasite. Nat Methods. 2011; 8(12):1078-82. PMC: 3431185. DOI: 10.1038/nmeth.1742. View

Deshmukh A, Srivastava S, Herrmann S, Gupta A, Mitra P, Gilberger T . The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing. Nucleic Acids Res. 2012; 40(12):5313-31. PMC: 3384324. DOI: 10.1093/nar/gks202. View

Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois A, Khim N . A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2013; 505(7481):50-5. PMC: 5007947. DOI: 10.1038/nature12876. View

Salanti A, Staalsoe T, Lavstsen T, Jensen A, Sowa M, Arnot D . Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria. Mol Microbiol. 2003; 49(1):179-91. DOI: 10.1046/j.1365-2958.2003.03570.x. View

Moon R, Hall J, Rangkuti F, Ho Y, Almond N, Mitchell G . Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A. 2012; 110(2):531-6. PMC: 3545754. DOI: 10.1073/pnas.1216457110. View

Kirkman L, Lawrence E, Deitsch K . Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity. Nucleic Acids Res. 2013; 42(1):370-9. PMC: 3874194. DOI: 10.1093/nar/gkt881. View

Mancio-Silva L, Rojas-Meza A, Vargas M, Scherf A, Hernandez-Rivas R . Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum. J Cell Sci. 2008; 121(Pt 12):2046-53. DOI: 10.1242/jcs.026427. View

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-23. PMC: 3795411. DOI: 10.1126/science.1231143. View

10.

Collins C, Das S, Wong E, Andenmatten N, Stallmach R, Hackett F . Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol. 2013; 88(4):687-701. PMC: 3708112. DOI: 10.1111/mmi.12206. View

11.

Duraisingh M, Triglia T, Cowman A . Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. Int J Parasitol. 2002; 32(1):81-9. DOI: 10.1016/s0020-7519(01)00345-9. View

12.

Gilbert L, Larson M, Morsut L, Liu Z, Brar G, Torres S . CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154(2):442-51. PMC: 3770145. DOI: 10.1016/j.cell.2013.06.044. View

13.

Bhaya D, Davison M, Barrangou R . CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet. 2011; 45:273-97. DOI: 10.1146/annurev-genet-110410-132430. View

14.

Crabb B, Cooke B, Reeder J, Waller R, Caruana S, Davern K . Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress. Cell. 1997; 89(2):287-96. DOI: 10.1016/s0092-8674(00)80207-x. View

15.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

16.

Bawankar P, Shaw P, Sardana R, Babar P, Patankar S . 5' and 3' end modifications of spliceosomal RNAs in Plasmodium falciparum. Mol Biol Rep. 2009; 37(4):2125-33. DOI: 10.1007/s11033-009-9682-4. View

17.

Wu Y, Sifri C, Lei H, Su X, Wellems T . Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci U S A. 1995; 92(4):973-7. PMC: 42619. DOI: 10.1073/pnas.92.4.973. View

18.

Deitsch K, Driskill C, Wellems T . Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res. 2001; 29(3):850-3. PMC: 30384. DOI: 10.1093/nar/29.3.850. View

19.

Straimer J, Lee M, Lee A, Zeitler B, Williams A, Pearl J . Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nat Methods. 2012; 9(10):993-8. PMC: 3697006. DOI: 10.1038/nmeth.2143. View

20.

Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J . RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-6. PMC: 3712628. DOI: 10.1126/science.1232033. View