Mutant/mutation
The mutant contains a DiCre expression cassette. The Dicre gene (the N-terminal Cre 59 (residues Thr19-Asn59) and C-terminal Cre 60 (Asn60-Asp343) portions of the Cre fused at their N-terminus to FKBP12 and FRB, respectively) is under control of the constitutive hsp70 promoter. The DiCre expression cassette is introduced into the silent p230p locus. In addition it expresses mCherry and does not contain a drug-selectable marker (see below).

The PyDiCre line was generated using the same construct as for P. berghei (RMgm-4900). This construct contains a DiCre cassette, as well as a mCherry cassette under the control of an inactive truncated fragment of HSP70 promoter, and a TgDHFR/TS pyrimethamine resistance cassette flanked by two LoxP sites, to allow Cre-mediated excision and production of drug selectable marker-free parasites. A sequence corresponding to the 3’ UTR of PbDHFR was included at the end of the construct, for homologous recombination at the modified p230p locus of drug selectable marker-free GFP expressing P. yoelii 17XNL parasites (RMgm-1028). Following transfection of PyGFP parasites, integration of the construct by double homologous recombination resulted in the replacement of GFP by mCherry and reconstitution of a functional HSP70 promoter to drive mCherry expression, along with insertion of the DiCre and TgDHFR/TS cassettes. Transfected parasites were selected with pyrimethamine and mCherry-positive parasites were sorted by FACS. The resulting parasite population was exposed to a single dose of rapamycin that was administered orally to mice. This treatment resulted in excision of the TgDHFR/TS cassette and retention of a single LoxP site. Cloning by limiting dilution resulted in the final drug selectable-marker free mCherry-expressing PyDiCre parasite line.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.11.14.623520

Protein (function)
The rapamycin-inducible Cre recombinase (DiCre) uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves. In the DiCre system, Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycine-?binding protein (either FKBP12 or FRB, the rapamycine-binding domain of the FKBP12 rapamycine-associated protein mTOR). Rapamycine-induced heterodimerization of the two components restores recombinase activity (rapamycine-?mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn-60, called Cre60) each fused to a different rapamycine-binding protein (FKBP12 and FRB respectively) results in the reconstitution of Cre recombinase activity).

This site–specific recombinase recognizes short, 34 bp sequences called loxP sites and catalyses the excision or inversion of the floxed (flanked by loxP) DNA segment.
Different systems have been developed to introduce DiCre into P. falciparum, including integration of DiCre cassettes into the chromosome and expression of the genes from an episome. Excision levels vary greatly between the systems, ranging from 50–98%. In these studies, parasite cultures were treated with 100 nM rapamycin for a minimum of four hours and high levels of excision of floxed DNA sequence were reported. However, both rapamycin and the carrier, DMSO, can negatively affect parasite growth at higher concentrations. 

Phenotype
From the paper: 
'Plasmodium yoelii S-type rDNA loci are dispensable in blood-stage parasites Prior investigations into S-type ribosome essentiality in P. berghei and P. yoelii demonstrated that sequences within S-type rDNA loci could be individually deleted in blood-stage parasites. The discrepancy between these studies exists in the phenotypes observed in mosquito-stage parasites with deletions of the S1 or S2 rDNA (also called the C- and D-loci, respectively), which could have arisen due to the different gene targeting strategies used. To address this apparent discrepancy and overcome past technical limitations, we leveraged a dimerizable Cre recombinase (DiCre) system in Plasmodium yoelii to first create clean deletions of the entire rDNA of either S-type ribosome. This system allows for the recycling of the selectable marker for pyrimethamine resistance, allowing us to then target the second S-type locus for subsequent deletion. To delete either the S1 (PY17X_0522400) or S2 (PY17X_0625700) rDNAs, we used conventional reverse genetics targeting upstream and downstream of the entirety of either S-type rDNA locus. The template provided for recombination contained a deletion cassette that conferred pyrimethamine resistance and constitutive GFP expression flanked by LoxP sites, further flanked by homology arms specific to either S1 or S2. Linearized plasmids were transfected into a P. yoelii line that had constitutive DiCre and mCherry expression. Similar to previous studies, we were able to readily delete the S-type rDNAs individually in blood-stage parasites. Transgenic parasites were selected for using pyrimethamine and confirmed by genotyping PCR, and enriched for using FACS for GFP+ and mCherry+ parasites. These enriched populations of transgenic parasites were then treated with rapamycin to excise the DNA encoding pyrimethamine resistance and GFP expression to generate parasites with clean deletions of the entire S1 or S2 rDNA locus, hereby called PyΔS1 or PyΔS2'

Additional information
DiCre recombinase mediated excision of targeted DNA sequences in vivo was achieved by injecting mice intraperitoneally with 4 mg/ml rapamycin (Fisher Scientific, Cat #AAJ62473MF) in 5% v/v PEG-400 (VWR, Cat # 200002-062), 5% v/v Tween-80 (VWR, Cat # VWRVM126-100ML), and 4% v/v DMSO (Fisher Scientific, Cat #PI85190) at a dosage of 4 mg rapamycin/kg mouse weight. Mice were injected with rapamycin when blood-stage parasitemia hit 1-2% and 48 hours later were euthanized by CO2 and exsanguinated by cardiac puncture for blood-stage parasite collection. Finally, transgenic parasites were cloned by limiting dilution approaches and clonality was validated by genotyping PCR.

Other mutants