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

Abstract

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...

Transcriptome-wide dynamics of extensive m6A mRNA methylation duringPlasmodium falciparumblood-stage development

“…Similarly, C-terminal tagging of the protein with a ligand-regulated Escherichia coli dihydrofolate reductase destabilization domain 39 for protein knockdown was unsuccessful. We therefore applied a genome editing-free method for gene down-regulation termed CRISPR interference (CRISPRi) 40 which has recently been adapted for Plasmodium 41,42 and is based on an enzymatically inactive (‘dead’) Cas9 protein (dCas9). In this method, the Cas9 protein is rendered inactive by two point mutations (Supplementary Fig.…”

“…As gene knockout was not possible, we used the recently developed CRISPRi system 41–43 to demonstrate the role of PfMT-A70 in the m 6 A methylation pathway. Because P. falciparum lacks the RNA interference pathway, application of CRISPRi is an invaluable new genome editing-free technique for targeted gene knockdown and functional characterization of essential genes in the parasite.…”

Cutting back malaria: CRISPR/Cas9 genome editing of Plasmodium

“…Our collective experience to date indicates that homology regions of >250–1000 bp are sufficient (Figure 1b), a similar range also observed by Ribeiro et al . [32], with efficient editing of P. yoelii with homology arms as short 80–100 bp as reported by Walker and Lindner [23]. However, to date there have been no reports of success using the very short homology regions (<50 bp) that are effective in Toxoplasma [40, 41].…”

“…Further developments in P. yoelii have included the production of a male/female reporter line expressing sex-enriched fluorescent proteins, as well as a parasite line constitutively expressing SpCas9 that would reduce the size of plasmids required [21, 22]. Recently, Walker and Lindner have reported the use of RNA polymerase II promoters to transcribe a ribozyme–guide–ribozyme system (CRISPR-RGR) in P. yoelii that can achieve high editing efficiencies for gene deletions and tag insertions [23]. This work also demonstrated that the number of gRNAs used influences gene editing outcomes, where using one gRNA can result in parasites bearing either plasmid integration and locus replacement gene edits, while using two gRNAs produces parasites with only locus replacement events.…”

“…Nonetheless, a recent study by Ribeiro et al [32] suggests that on-target scores are predictive of success even in P. falciparum , and report an annotated list of all 662 795 potential gRNA sites in the P. falciparum genome. Another consequence of AT-rich genomes is the potential for poly-T stretches within the gRNA sequence, which could result in premature termination by RNA pol III and T7 RNAP, and thus should be avoided unless an RNA pol II expression system, like CRISPR-RGR, is used [23, 33]. Finally, recent ATAC-seq data on P. falciparum [34, 35] may be useful to inform gRNA selection to bias towards open chromatin or to troubleshoot unsuccessful editing events, as chromatin accessibility has been shown to affect editing in other systems [36].…”