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. Importance Plasmodium 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.


Introduction
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 reverse genetic techniques 40 to disrupt or tag genes-of-interest, however, producing gene modifications in Plasmodium 41 parasites has inherent challenges. First, transfection efficiencies are surprisingly low compared to CRISPR/SpCas9 gene editing systems function by expressing sgRNAs (single guide RNAs) that 67 recruit the Streptococcus pyogenes Cas9 (SpCas9) endonuclease to a complementary 20nt 68 sequence of genomic DNA. There, a double-stranded break (DSB) is created and typically 69 repaired with either the error-prone Non-Homologous End Joining (NHEJ) or by Homology- 70 Directed Repair (HDR) pathways. Because Plasmodium parasites lack several essential proteins 71 required for NHEJ, homology-directed repair of double-strand breaks predominates, with 72 infrequent repair also occurring by microhomology-mediated end joining (MMEJ) (16,17). 73 Therefore, strategies for CRISPR gene editing in Plasmodium require the introduction of three 74 components: SpCas9, sgRNAs, and a homology directed repair (HDR) template, which are 75 typically encoded on one or two nuclear plasmids. 76 CRISPR-based gene editing of P. falciparum has been achieved using dual-plasmid systems, 77 with each plasmid encoding a unique drug resistance marker, and either SpCas9 or the HDR and 78 sgRNAs (11,12). Efficient expression of sgRNAs was demonstrated with either an engineered 79 T7 RNA polymerase system or with the RNA polymerase III transcribed P. falciparum U6 80 promoter (11,12). Upon electroporation, parasites are then pressured with one or both drugs 81 simultaneously to select for parasites containing all of the necessary gene editing elements (11,82 12). 83 CRISPR/Cas9 strategies in P. yoelii, however, are limited by the availability of only one drug-84 selectable marker (DHFR), so all CRISPR/SpCas9 gene editing elements must be packaged onto 85 a single vector to allow their selection. Previously described single-plasmid systems use the P. 86 yoelii U6 promoter to express sgRNAs, however the resulting transgenic parasites were found to 87 retain the plasmid sequences and remained resistant to drug pressure (13). Because of this, a 88 second system was constructed that included a negative-selectable marker in the plasmid 89 5 backbone, so that parasites that retained a nuclear plasmid or that may have integrated the 90 plasmid could be selected against following gene editing (18). Despite these limitations, the 91 laboratory of Jing Yuan has used CRISPR to methodically interrogate the ApiAP2 gene family 92 and genes important for ookinete motility, and have created transgenic parasites that express a 93 constitutively expressed SpCas9 nuclease, or male and female-enriched fluorescent protein 94 reporters (13,(18)(19)(20)(21). 95 Although significant progress has been made to date in the development of CRISPR tools for use 96 in Plasmodium, significant limitations remain. First, existing systems use RNAP III promoters 97 for sgRNA expression, which is preferred due to the well-defined 5' and 3' ends on this class of 98 transcript. However, as RNAP III promoters are strong and constitutively active as required to 99 produce 5S rRNA, tRNAs and other critical non-coding RNAs, their use in transcribing sgRNAs 100 would not permit stage-specific or readily tunable expression. Secondly, most studies to date 101 have targeted a single locus with one sgRNA, and those few efforts that have used multiple 102 sgRNAs used multiple RNAP III-based cassettes to express the sgRNAs. Lastly, the adoption 103 and use of nuclease-dead variants of SpCas9 (dSpCas9), which has been used in prokaryotes and 104 eukaryotes for gene regulation by CRISPR activation (CRISPRa) and CRISPR interference 105 (CRISPRi), has not been demonstrated. Because Plasmodium parasites lack genes essential for 106 RNA interference (RNAi) (22), genetic tools to regulate transcript abundance (e.g. promoter 107 swap, glmS) or protein abundance (e.g. DD/Shield1, EcDHFR-DD/Trimethoprim, TetR/DOZI) 108 have been developed and utilized (23)(24)(25)(26). However, these all require single or multiple 109 modifications to the genome and introduce exogenous sequences into the locus-of-interest.

110
CRISPRi is therefore a desirable tool, as it would allow for regulation of the expression of 111 specific genes without modification of the genome itself. Here, we show that CRISPR-RGR is able to effectively generate gene deletions, tag insertions, 113 and can be used for CRISPR interference. Using a Ribozyme-Guide-Ribozyme (RGR) method of 114 sgRNA expression, we show that three RNAP II promoters can be used to express multiple 115 sgRNAs simultaneously, and that genome editing can be rapidly and efficiently achieved in P. 116 yoelii. Additionally, we demonstrate that the number of sgRNAs used to target a gene influences 117 the outcome of genome repair, and that negative selection is not required to produce parasites 118 with only locus replacement events observed. Finally, we demonstrate that CRISPRi is possible 119 in P. yoelii, with the efficacy of 11 individual sgRNAs targeting across the upstream portion of 120 the gene control region of PyALBA4 demonstrating positional, but not strand-specific, effects.

121
The contribution of CRISPR-RGR to the growing Plasmodium CRISPR toolbox explains and 122 solves previous issues with current strategies that use one sgRNA, improves how synthetic 123 biology approaches can be used to expedite gene editing, and provides a framework for 124 CRISPRi-based gene regulation. To streamline CRISPR/SpCas9 editing in Plasmodium yoelii parasites, we developed a flexible, 133 single-plasmid construct that contains all necessary CRISPR/SpCas9 gene editing elements ( Fig.   134 1, 1 st Generation). Expression of SpCas9::GFP, HsDHFR (to provide resistance to anti-folate 135 drugs), and sgRNAs were generated by individual iterations of the strong, constitutive pbef1α 136 promoter and the pbdhfr 3'UTR/terminator. Each of these cassettes is flanked by unique 137 restriction enzyme sites for easy modification and substitutions. In addition to these cassettes, we 138 incorporated a homology-directed repair (HDR) template to enable homology directed repair of 139 the double-strand breaks (DSBs) that are created by SpCas9. Because the sequence conservation 140 of these gene control elements between P. yoelii and P. berghei is exceptionally high, and that in 141 fact a mixture of elements from both species are used in the plasmids describe here, we 142 anticipate that gene editing using these plasmids will be possible in P. berghei as well.

144
The major differences between this CRISPR-based editing system for Plasmodium and those 145 previously described lie in the expression of the sgRNAs and the preparation of the sgRNA 146 and HDR template sequences. Existing Plasmodium CRISPR systems use RNA Polymerase 147 III-driven U6 promoters or T7 RNA Polymerase-based systems for sgRNA expression (11,148 12). In contrast, we have expressed a transcript encoding a Ribozyme-Guide-Ribozyme (RGR) 149 unit that uses a minimal Hammerhead Ribozyme and a Hepatitis Delta Virus ribozyme to flank 150 the sgRNA on its 5' and 3' ends, respectively (Fig. 1, inset). This RGR approach, first 151 described in yeast (27) and since used in Leishmania (28) and zebrafish (29), generates 152 sgRNAs with precisely defined 5' and 3' ends, and allows for the simultaneous expression of shown to have increase editing efficiency (30). Finally, we leverage advances in synthetic 165 biology to create custom DNA fragments that can include the RGR transcript, short HDR 166 templates, or both, which greatly expedites plasmid generation. We anticipate that as the cost 167 of gene synthesis continues to decrease, these approaches can be scaled for use in both forward 168 and reverse genetic screens. A step-by-step tutorial for construct generation is provided in  In order to functionally test this single-plasmid, CRISPR-RGR system, we targeted the gene 173 encoding the PyALBA4 RNA-binding protein, which we have previously characterized (10).

174
Using conventional reverse genetics approaches, we have shown that pyalba4 can be deleted in 175 asexual blood stage parasites, which results in the production of 2-3 fold more mature male 176 gametocytes that can exflagellate as compared to wild-type parasites. Additionally, a C-terminal 177 GFP tag can be introduced with no observable effect upon parasite growth or transmission. In 178 order to delete pyalba4 by CRISPR-RGR, two sgRNA targets were chosen at the 5' and 3' ends 179 of its coding sequence by manually scanning for NGG PAM motifs and subsequent 180 computational assessment using the Eukaryotic Pathogen CRISPR guide RNA/DNA design tool 181 (http://grna.ctegd.uga.edu) ( Fig. 2A, red vertical lines). This tool provides a score for each 182 sgRNA based on the target specificity within the genome, as well the GC content and position-183 specific nucleotide composition for bases that have been shown to affect sgRNA efficiency. Because CRISPR-RGR rapidly and efficiently produced transgenic parasites when providing 211 large homology arms in the HDR template, we tested the effect that lengthening (~1000bp each 212 arm) and shortening (~80-100bp, ~250bp each arm) the homology arms had upon gene editing. 213 We observed that all homology arm lengths allowed for efficient gene editing, and that the 214 smallest HDR tested (80-100bp each arm) had the most efficient editing (as evidenced by the 215 least intense PCR amplicons for wild-type parasites) and could be selected in the same amount of 216 time as was required for the longer HDR templates (Fig. 2C). Importantly, HDR arms of this 217 length, even with the skewed A-T content of P. yoelii's genome, can be chemically synthesized. 218 It is notable that over the course of these experiments, we observed that recombination was 219 occurring in E. coli between two instances of the pbef1α promoter, and that the RGR portion of  transgenic parasites created by conventional methods (Fig. 3C).

242
In contrast to conventional reverse genetic techniques, CRISPR/SpCas9 gene editing can be 243 accomplished without leaving a drug selectable marker in the edited locus. This enables multiple, 244 sequential gene edits to be made after curing the delivery plasmid, which is particularly useful 245 with rodent-infectious malaria parasites where only one drug selectable marker (DHFR) is 246 available. However, previous work has shown that completely curing these plasmids is 247 challenging, as resistant parasites can be recovered more than 50 days post-removal of drug 248 pressure (18). To circumvent this issue, methods have been developed to negatively select 249 against parasites that retained the delivery plasmid following genome modification (18). We 250 similarly attempted to cure the plasmid from FACS-selected PyALBA4::GFP parasites produced 251 using one sgRNA, and observed that the parasites remained drug resistant after more than 14 252 days following removal of drug pressure. An expanded genotyping PCR assay that interrogates 253 for both plasmid integration and locus replacement showed that both editing outcomes occurred 254 (Fig. 3B). 255 We reasoned that introduction of two DSBs, and thus two genome repair events, would ensure 256 that only locus replacement events would result. To test this, we selected a second sgRNA target 257 in the pyalba4 3' UTR downstream of the original sgRNA target, and introduced a second shield 258 mutation into the HDR template (Fig. 3A). Transfection of this plasmid, coupled with constant 259 selection with pyrimethamine, produced PyALBA4::GFP-expressing parasites. As before, 260 genotyping PCR showed that a significant fraction of the parasite population had been modified.

261
Notably, this two sgRNA design yielded only the desired locus replacement events and showed 262 no evidence of plasmid integration (Fig. 3B, right). Furthermore, upon removal of drug pressure, 263 the parasites quickly (<1 week) became sensitive to pyrimethamine once more and were 264 amenable to subsequent transfections.

279
For this approach, we constructed a CRISPRi plasmid similar to our 1 st generation CRISPR 280 plasmid but without an HDR template, which is unnecessary for this application (Fig. 1). This Using RNA-seq data to estimate the 5' UTR of the PyALBA4 locus (Fig. 4A, Supp. Fig. 3), we 293 selected eleven individual sgRNA targets between the start of the contiguous RNA-seq reads 294 (approximately -800) and the ATG. We selected sgRNAs that will target either the template or 295 non-template strands of DNA to assess if strand-specific effects occur in Plasmodium, and that 296 target DNA at various distances away from the ATG to assess positional effects (Fig. 4A). The  To assess whether we could impose CRISPRi regulation upon this locus, we used flow 304 cytometry to monitor PyALBA4::GFP protein abundance. This was measured in 305 dSpCas9::1xHA-positive parasites expressing one of the eleven on-target sgRNAs or the no 306 target sgRNA control that have the capacity for CRISPRi (Fig. 4, Supp Fig. 2). Parasites were 307 synchronized to the schizont stage to minimize the effect of stage-specific variance in 308 PyALBA4::GFP expression upon these observations. Parasites with background levels (negative Here we present CRISPR-RGR, a ribozyme-based CRISPR system for Plasmodium yoelii that 332 allows rapid and efficient gene editing in rodent-infectious parasites. This approach is powerful 333 and provides advantages over current methods. First, the production of multiple sgRNAs from a 334 single transcript greatly reduces the potential size of plasmids required for CRISPR-based gene 335 editing by using a single promoter and terminator for sgRNA expression. Importantly, these 336 sgRNAs can be designed to target a single gene in multiple locations as used here, but can be 337 extended to target multiple genes. The utility of using multiple sgRNAs for a single gene is 338 evident from the data presented here: the use of one sgRNA can result in a mixture of gene 339 editing outcomes (plasmid integration and locus replacement), whereas the use of two sgRNAs 340 produced only the desired locus replacement result. We strongly suggest that two or more 341 sgRNAs be used for CRISPR-based gene editing where locus replacement is the desired outcome 342 to eliminate the need for negative selection. Second, CRISPR-RGR can be programmed using 343 synthetically produced DNA fragments that can encode the RGR, HDR, or both elements, which 344 can be inserted in one or two molecular cloning steps. With the anticipated decreases in cost and 345 increases in capacity to synthesize large DNA fragments, this approach will streamline reagent 346 preparation and enable CRISPR screens at scale. Lastly, the FACS-based selection of transgenic 347 parasites expressing SpCas9::GFP or a protein-of-interest fused to GFP reduces the number of 348 mice required to produce transgenic parasite lines free from observable wild-type parasites.

349
Because it is still possible that wild-type parasites remain in the population at levels lower than 350 the limit of detection of PCR, caution is urged when phenotyping these parasites and it is our 351 opinion that these parasites should be cloned prior to these studies.      Table 1). An empty cassette consisting of the pbef1α promoter and pbdhfr3'UTR were  Table 2). These products were inserted into and sequenced in 460 pCR-Blunt, and then subcloned into pSL1165 to create pSL1166 (using the pybip promoter) and 461 pSL1211 (using the pygapdh promoter). These plasmids were transfected into Plasmodium yoelii 462 17XNL strain parasites and were analyzed for their editing efficiency. The empty vector and 463 pyalba4-targeted plasmids described in this work will be available on Addgene.  Table   469 1). Accudenz density gradient (2,40), and were prepared identically to IFA samples as described      Table 1.   Table 1: Oligonucleotide sequences used in this study. 590 These sequences include primers used to amplify the CRISPR plasmid elements, such as the 591 SpCas9, promoter sequences, and the HDR templates used, as well as those used for genotyping 592 PCR of the transgenic parasites that were generated.   Table 3: Nucleotide sequences and positions of sgRNAs used in this study 600 Each 20nt sequence of the sgRNA targets used in this study are annotated with their position and 601 the experiments they were used for.     Total RNA-seq (Py17XNL)