Plasmid-free CRISPR/Cas9 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733Emily D. Crawford, Jenai Quan, Jeremy A. Horst, Daniel Ebert Wesley Wu, Joseph L. DeRisi
PLoS One, 2017Abstract: 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.