Creation of Cas9-expressing Plasmodium falciparum

The current CRISPR/Cas9 system for Plasmodium falciparum has technical problems caused by plasmid constructs, such as delay in establishing transgenic parasites during drug selection and unexpected integration of circular donor DNA by single-crossover recombination. Although these problems can be solved by using linear donor templates, it requires highly efficient introduction of DNA and fast completion of recombination because linear DNA is easily lost from parasites during multiplication. Here, we overcame those problems by developing a highly efficient DNA transfer method and Cas9-expressing parasites. Using our new CRISPR/Cas9 system, transgenic parasites were established in two weeks without any unexpected recombination nor off-target mutations. Furthermore, by our system, two genes on different chromosomes were successfully modified in one transfection. Because of its high efficiency and robustness, our new CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum and dramatically accelerate the studies.