1Functional characterization of genes in Plasmodium parasites often relies on genetic 2 manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by 3 the time required to generate transgenic parasites for P. falciparum and the availability of a 4 single drug selectable marker for P. yoelii. In both cases, there remains a risk of disrupting native 5 gene regulatory elements with the introduction of exogenous sequences. To address these 6 limitations, we have developed CRISPR-RGR, a SpCas9-based gene editing system for 7 Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. 8Using this system with P. yoelii, we demonstrate that both gene disruptions and coding sequence 9 insertions are efficiently generated, producing marker-free and scar-free parasites with homology 10 arms as short as 80-100bp. Moreover, we find that the common practice of using one sgRNA can 11 produce both unintended plasmid integration and the desired locus replacement editing events, 12 while the use of two sgRNAs results in only locus replacement editing. Lastly, we show that 13 CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding dCas9 to targets in 14 the gene control region of a gene-of-interest, resulting in a modest reduction in gene expression. 15 This robust and flexible system should open the door for in-depth and efficient genetic 16 characterizations in both rodent-and human-infectious Plasmodium species. 17 18 Importance 19Plasmodium parasites, the causative agent of malaria, still pose an enormous threat to public 20 health worldwide. Gaining additional insight into the biology of the parasite is essential for 21 generating an effective vaccine and identifying novel drug targets. To this end, CRISPR/Cas9 22 23 possible with conventional reverse genetics approaches. Here, we describe CRISPR-RGR as an 24 addition to the CRISPR/Cas9 toolbox for the rodent -infectious Plasmodium parasites. By using 25 multiple ribozyme-flanked single guide RNAs expressed from RNA polymerase II promoters, 26 transgenic parasites can be rapidly generated as designed without leaving selectable markers. 27Moreover, CRISPR-RGR can be adapted for use as a CRISPR interference (CRISPRi) system to 28 alter gene expression without genome modification. Together, CRISPR-RGR for gene editing 29 and CRISPRi application can hasten investigations into the biology and vulnerabilities of the 30 malaria parasite.31 32 33 Introduction 34 Malaria remains one of the world's most daunting public health concerns, with over 200 million 35 infections and nearly half a million fatalities every year (WHO, 2017). Despite gains made to 36 reduce transmission worldwide, there is still a need for a highly effective, licensed vaccine and 37 additional anti-malarial drugs to respond to and overcome the emergence and spread of drug 38 resistance. To produce these new therapeutics, further studies of the causal agent of malaria, the 39 Plasmodium parasite, are required. These studies typically rely upon re...
40 SUMMARY STATEMENT 41A gain-of-function screen of the human kinome revealed AAK1 as a negative regulator of WNT 42 signaling. We show that AAK1 promotes clathrin-mediated endocytosis of LRP6, resulting in 43 downregulation of WNT signaling. We use a new selective and potent AAK1/BMP2K small 44 molecule probe to validate our findings.
19With relatively few known specific transcription factors to control the abundance of specific 20 mRNAs, Plasmodium parasites also regulate the stability and turnover of transcripts to provide 21 more comprehensive gene regulation. Plasmodium transmission stages impose translational 22 38 39 3 AUTHOR SUMMARY 40 Malaria is a disease caused by Plasmodium parasites, which are transmitted during an 41 infectious blood meal by anopheline mosquitoes. Transmission of the sexual stages of the 42 parasite to mosquitoes requires the proper regulation of specific mRNAs. While much work has 43 been done to characterize regulation of mRNAs in female gametocytes, little has been done to 44 assess this regulation in male gametocytes. Here, we demonstrate that PyCCR4-1, a member of 45 the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly 46 in both male and female parasites, and results in a reduction of male gametocytemia. In 47
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