Mutant/mutation
In the mutant the alba4 gene has been disrupted using a newly developed CRISPR-RGR, a Streptococcus pyogenes (Sp) Cas9-based gene editing system for Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) single-guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. see 'Additional information' below for more information.

Protein (function)
ALBA proteins are evolutionarily conserved from Archaea and consistently have been found to bind to nucleic acids. Structurally, the ALBA domain resembles the IF3 C-terminal domain, which binds to the small subunit of the prokaryotic ribosome. Together, this strategy provides a straightforward mechanism for ALBA proteins to tether nucleic acids to the ribosome. Plasmodium spp. contain at least four ALBA-domain containing proteins, and have been shown to bind both DNA and RNA. Of these ALBA proteins, ALBA4 is specific to the Apicomplexan lineage and its Chromerid ancestor (40-50%; identity / 60-65% similarity).

Phenotype
In order to functionally test this single-plasmid, CRISPR-RGR system, we targeted the gene encoding the PyALBA4 RNA-binding protein, which we have previously characterized. Using conventional reverse genetics approaches, we have shown that pyalba4 can be deleted in asexual blood stage parasites, which results in the production of 2-3 fold more mature male gametocytes that can exflagellate as compared to wild-type parasites. Additionally, a C-terminal GFP tag can be introduced with no observable effect upon parasite growth or transmission. In order to delete pyalba4 by CRISPR-RGR, two sgRNA targets were chosen at the 5’ and 3’ ends of its coding sequence by manually scanning for NGG PAM motifs and subsequent computational assessment using the Eukaryotic Pathogen CRISPR guide RNA/DNA design tool (http://grna.ctegd.uga.edu). This tool provides a score for each sgRNA based on the target specificity within the genome, as well the GC content and position-specific nucleotide composition for bases that have been shown to affect sgRNA efficiency. Additionally, this tool will flag any sgRNA with long poly-T tracts (more than four in a row) which can cause early termination of RNA polymerase III transcripts. Because the RGR system utilizes RNA polymerase II promoters (e.g. pbef1α, pygapdh, pybip), we are not limited by sgRNAs containing poly-T tracts that are prevalent in Plasmodium genomes. For the first trials of our system, we chose to use the pbef1α promoter to drive expression of the RGR. The initial HDR template was designed with homology arms comprised of ~800bp of sequence homologous to the target gene on either side of the two DSBs, with a unique 18bp DNA barcode between them that could be used for unambiguous, simple genotyping PCR. Notably, this barcode is not necessary for genome editing and could be omitted to produce completely scar-less modifications without the introduction of any exogenous sequences.

Upon transfection of this plasmid into wild type (WT) Plasmodium yoelii (17XNL strain) parasites with constant pyrimethamine selection, mice reached 1% parasitemia in 8 days, which is a similar timeframe to conventional techniques. We observed that a large subset of these parasites showed expression of SpCas9::GFP by live fluorescence microscopy, and genotyping PCR analysis showed efficient editing of the pyalba4 locus. Furthermore, by enriching for SpCas9::GFPpositive schizonts via Fluorescence-activated Cell Sorting (FACS), only edited parasites were present by genotyping PCR. Thus, this CRISPR-RGR
approach rapidly produced a transgenic parasite population with no observable wild-type parasites present using as few as two mice.

We further verified that this population of CRISPR-generated pyalba4- parasites had the same phenotype as pyalba4- transgenic parasites generated by conventional reverse genetic approaches. Quantification of the activation of male gametocytes into gametes (measured as centers-of-movement/exflagellation centers via light microscopy) in the pyalba4- line revealed a similar 2-3 fold increase in the number of activated male gametocytes as compared to wild type parasites, which was sustained across the full duration of the infection. Because CRISPR-RGR rapidly and efficiently produced transgenic parasites when providing large homology arms in the HDR template, we tested the effect that lengthening (~1000bp each arm) and shortening (~80-100bp, ~250bp each arm) the homology arms had upon gene editing. We observed that all homology arm lengths allowed for efficient gene editing, and that the smallest HDR tested (80-100bp each arm) had equally efficient editing (as evidenced by the least intense PCR amplicons for wild-type parasites) and could be selected in the same amount of time as was required for the longer HDR templates. Importantly, HDR arms of this length, even with the skewed A-T content of the P. yoelii genome, can be chemically synthesized.

It is notable that over the course of these experiments, we observed that recombination was occurring in E. coli between two instances of the pbef1α promoter, and that the RGR portion of the plasmid was being excised. To stabilize the plasmid, we constructed a second generation of editing plasmids with no repeated elements and have not observed spurious recombination events occurring with this new design (2nd Generation). This second generation design includes a single iteration of the pbef1α promoter and pyef1α 3’UTR to control expression of HsDHFR, the pydhfr promoter and pbdhfr 3’UTR to control expression of SpCas9::GFP, and either the pybip promoter or pygapdh promoter with the pybip 3’UTR to control transcription of the RGR element. Using the same sgRNA targets and the 191bp HDR template, we found that both promoters driving RGR expression edited parasites efficiently. Furthermore, we again verified with FACS and genotyping PCR that parasites expressing SpCas9::GFP only contained the edited pyalba4 locus. Together these results show that single plasmid CRISPR-RGR is able to efficiently and robustly create gene deletions in P. yoelii parasites.

Additional information
Although significant progress has been made to date in the development of CRISPR tools for use in Plasmodium, significant limitations remain. First, most existing systems use RNA polymerase III promoters for sgRNA expression, which is preferred due to the well-defined 5’ and 3’ ends on this class of transcript. However, as RNA polymerase III promoters are strong and constitutively active as required to produce 5S rRNA, tRNAs and other critical non-coding RNAs, their use in transcribing sgRNAs would not permit stage-specific or readily tunable expression. In systems outside Plasmodium, tools have been generated to produce multiple sgRNAs under the control of a single RNA polymerase II promoter. The Ribozyme-guide-Ribozyme (RGR) and the miRNA polycistron tools both utilize post-transcriptional modifications to an RNA transcript to generate pristine sgRNAs.

Here, we show that CRISPR-RGR (Ribozyme-Guide-Ribozyme) is able to effectively generate gene deletions, tag insertions, sequential genome editing.

Single-plasmid, ribozyme-mediated CRISPR/SpCas9 plasmid design for rodent infectious Plasmodium parasites
To streamline CRISPR/SpCas9 editing in Plasmodium yoelii parasites, we developed a flexible, single-plasmid construct that contains all necessary CRISPR/SpCas9 gene editing elements using a combination of P. yoelii and P. berghei promoters and terminators (1st Generation). Expression of SpCas9::GFP, HsDHFR (to provide resistance to anti-folate drugs), and sgRNAs were generated by individual iterations of the strong, constitutive pbef1α promoter and the pbdhfr 3’UTR/terminator. Each of these cassettes is flanked by unique restriction enzyme sites for easy modification and substitutions. In addition to these cassettes, we incorporated a homology directed repair (HDR) template to enable homology directed repair of the double-strand breaks (DSBs) that are created by SpCas9.

The major differences between this CRISPR based editing system for Plasmodium and those previously described lie in the method of expression of the sgRNAs and the preparation of the sgRNA and HDR template sequences. Existing Plasmodium CRISPR systems use RNA polymerase III-driven U6 promoters or T7 RNA polymerase-based systems for sgRNA expression. In contrast, we have expressed a transcript encoding a Ribozyme-Guide-Ribozyme (RGR) unit that uses a minimal Hammerhead Ribozyme and a Hepatitis Delta Virus ribozyme to flank the sgRNA on its 5’ and 3’ ends, respectively. This RGR approach, first described in yeast and since used in Leishmania and zebrafish, generates sgRNAs with precisely defined 5’ and 3’ ends, and allows for the simultaneous expression of multiple guides under control of a single promoter, including RNA  polymerase II promoters.

While RNA polymerase II promoters are far more abundant than RNA polymerase III promoters, they are not typically used for sgRNA expression as their transcripts can initiate from multiple transcriptional start sites (TSS’s) and are capped and polyadenylated. The potential impact of these 5’ extensions and modifications upon sgRNA activity are not sufficiently understood to confidently use them for this application. However, the inclusion of autocatalytic, self-cleaving ribozymes within an RNA eliminates these potential problems with RNA polymerase II transcripts, and their beneficial properties of stage-specific and tunable expression levels can be used. Moreover, these individual RGR units can be polymerized into an RGR array on a single transcript and be used to generate multiple sgRNAs. In addition, our sgRNA design contains an extended duplex and a single base change compared to the original sgRNA sequence, which has been shown to have increased editing efficiency.

It is notable that over the course of these experiments, we observed that recombination
was occurring in E. coli between two instances of the pbef1α promoter, and that the RGR portion of the plasmid was being excised. To stabilize the plasmid, we constructed a second generation of editing plasmids with no repeated elements and have not observed spurious recombination events occurring with this new design (2nd Generation). This second generation design includes a single iteration of the pbef1α promoter and pyef1α 3’UTR to control expression of HsDHFR, the pydhfr promoter and pbdhfr 3’UTR to control expression of SpCas9::GFP, and either the pybip promoter or pygapdh promoter with the pybip 3’UTR to control transcription of the RGR element. Using the same sgRNA targets and the 191bp HDR template, we found that both promoters driving RGR expression edited parasites efficiently. Furthermore, we again verified with FACS and genotyping PCR that parasites expressing SpCas9::GFP only contained the edited pyalba4 locus. Together these results show that single plasmid CRISPR-RGR is able to efficiently and robustly create gene deletions in P. yoelii parasites.

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