Mutant/mutation
In the mutant the alba4 gene has been C-terminally tagged with GFP 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 (and see also mutant RMgm-4632).

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
We used CRISPR-RGR (see below and mutant RMgm-4632) to append a C-terminal GFP epitope tag to PyALBA4. To accomplish this, we chose a single sgRNA target (1x sgRNA) 18bp downstream of the stop codon and designed a HDR template with ~700bp homology arms flanking an in-frame GFP tag. A base change of the NGG PAM sequence (shield mutation) was included in the HDR template so that SpCas9 ceases to bind and cut this locus post-editing. These modifications were built into the first generation CRISPR plasmid with the pbef1α promoter driving sgRNA expression. Transfection of this plasmid, along with constant pyrimethamine selection for 7 days, yielded a largely transgenic parasite population with PyALBA4::GFP expression matching what was seen in transgenic parasites created by conventional methods (RMgm-4314). Expression has not been analysed in detail: GFP expression in cytoplasm of schizonts.

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.

Other mutants
see also RMgm-4632 for a mutant in which the alba4 gene has been disrupted using this newly developed CRISPR-RGR system