Plasmid-free CRISPR/Cas9 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733

Crawford ED1,2,3, Quan J1,2,3, Horst JA2, Ebert D2,3, Wu W2, DeRisi JL1,2,3.
Source: PLoS ONE
Publication Date: (2017)
Issue: 12: 5
Research Area:
Drug Discovery
Cells used in publication:
Plasmodium falciparum
Species: unicellular
Tissue Origin:
96-well Shuttleâ„¢ System
4D-Nucleofectorâ„¢ X-Unit

High parasitemia (17% of RBCs infected with parasites), synchronized ring-stage parasite cultures grown in fresh donor red blood cells at 2% hematocrit were transfected in 96-well Lonza Nucleofector plates (Lonza, Basel, Switzerland). Transfection was done on the Lonza 96-well shuttle on pulse setting CM-162


Genetic manipulation of the deadly malaria parasite Plasmodium falciparum remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite's genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in P. falciparum without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in P. falciparum could greatly simplify future editing attempts regardless of the nuclease used or the delivery method.