Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System

Abstract

Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites.

Current therapies and future possibilities for drug development against liver-stage malaria

“…Functional genomic tools such as RNA interference and the CRISPR-Cas9 genome editing system will greatly benefit efforts to identify possible host targets and map host-parasite interactions, both of which are compatible with genome-wide screening. Additionally, the CRISPR-Cas9 system can target the Plasmodium parasite (102)(103)(104), facilitating studies to validate parasite targets. Renowned for its simplicity and versatility, CRISPR-Cas9 offers unique opportunities (e.g., introduction of point mutations, gene knockout) to investigate key host-parasite interactions during liver-stage malaria in vitro, and most importantly in vivo.…”

Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker

“…Similarly, sinefungin-resistant lines have been made in Plasmodium falciparum (35), but the gene responsible for resistance remains unidentified. With the advent of CRISPR technology in Plasmodium (36)(37)(38), transporters with homology to either the Leishmania AdoMet1 (32) or T. gondii SNR1 genes can be disrupted and tested for whether they confer SNF r , possibly providing a useful selectable marker.…”

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

“…Recent reports in both P. falciparum (172) and P. yoelii (173) have successfully shown the ability of the CRISPR/Cas system to introduce SNPs into a gene of interest, tag proteins (e.g., with gfp), and knock out coding sequence with and without a marker (e.g., human dhfr). The outcomes of each experiment showed a high editing efficiency with no detectable amount of undesirable, off-target events.…”

“…The first uses of the CRISPR/Cas system in Plasmodium research have only recently been published (172,173). This system modifies a prokaryotic viral defense system to cleave a specific genomic sequence harboring a unique motif, using an RNA-guided Cas9 endonuclease (164)(165)(166).…”