Abstract
Malaria is caused by infection with Plasmodium parasites and is a major public health concern. The CRISPR/Cas9 system is a promising technology, but still has technical problems, such as low efficiency and unexpected recombination. Here, we solved these problems by transfecting Cas9-expressing parasites with linear donor templates. The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large-scale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the post-genomic era of Plasmodium species.