Ribozyme-Mediated, Multiplex CRISPR Gene Editing and CRISPRi in Plasmodium yoelii

Functional characterization of genes in Plasmodium parasites often relies on genetic manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by the time required to generate transgenic parasites for P. falciparum and the availability of a single drug selectable marker for P. yoelii. In both cases, there remains a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a SpCas9-based gene editing system for Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. Using this system with P. yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free and scar-free parasites with homology arms as short as 80-100bp. Moreover, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and the desired locus replacement editing events, while the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding dCas9 to targets in the gene control region of a gene-of-interest, resulting in a modest reduction in gene expression. This robust and flexible system should open the door for in-depth and efficient genetic characterizations in both rodent- and human-infectious Plasmodium species.\n\nImportancePlasmodium parasites, the causative agent of malaria, still pose an enormous threat to public health worldwide. Gaining additional insight into the biology of the parasite is essential for generating an effective vaccine and identifying novel drug targets. To this end, CRISPR/Cas9 tools have been developed to more efficiently interrogate the Plasmodium genome than is possible with conventional reverse genetics approaches. Here, we describe CRISPR-RGR as an addition to the CRISPR/Cas9 toolbox for the rodent-infectious Plasmodium parasites. By using multiple ribozyme-flanked single guide RNAs expressed from RNA polymerase II promoters, transgenic parasites can be rapidly generated as designed without leaving selectable markers. Moreover, CRISPR-RGR can be adapted for use as a CRISPR interference (CRISPRi) system to alter gene expression without genome modification. Together, CRISPR-RGR for gene editing and CRISPRi application can hasten investigations into the biology and vulnerabilities of the malaria parasite.