Mutant/mutation
The mutant expresses both DiCre and Cas9. Both genes are introduced into the silent p230p gene locus and are under control of constitutive promoters. In addition, the mutant expresses GFP under control of the constitutive hsp70 promoter. Parasites of this line does not contain a drug-selectable marker cassette.
The DiCre expression cassette is introduced in the transgenic P. berghei PbCas9 line containing a Cas9 cassette integrated in the dispensable p230p locus under the control of the hsp70 promoter for constitutive expression. This PbCas9 line was generated similar to the transgenic P. berghei line containing the cas9 expression cassette introduced into the c/d-ssr-rna gene locus (RMgm-4870). Parasites of this line does not contain a drug-selectable marker'.
In order to combine the Cas9 and DiCre systems, we used CRISPR to genetically modify PbCas9 parasites. A 20-base sgRNA target was selected in the p230p locus, downstream of the integrated Cas9 cassette, and the corresponding sequence was cloned downstream of PbU6 promoter into a sgRNA plasmid containing a pyrimethamine-resistance cassette. For the repair DNA template, we used the same DiCre cassette as previously described (see RMgm-4900). This cassette consists in 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. These two components were placed under control of the constitutive bidirectional promoter eef1α, and followed by the 3’ untranslated region (UTR) from Pfcam and Pbhsp70, respectively. In this construct, a fluorescent marker (GFP or mCherry, respectively) was incorporated under the control of the hsp70 promoter and the 3’ UTR of Pbdhfr. On each side of the construct, 5’ and 3’ homology fragments were intgrated for integration at the p230p locus, downstream of the Cas9 cassette. The donor template DNA was assembled in a plasmid and linearized prior to transfection.
The recombinant parasite lines were exposed to 5-fluorocytosine (5-FC) to eliminate any residual parasite harboring the sgRNA plasmid, resulting in pure, drug-selectable marker free PbCasDiCre-GFP and PbCasDiCre-mCherry parasite populations.

Owing to the dual integration of CRISPR-Cas9 and DiCre, these transgenic parasites allow unparalleled levels of gene modification and conditional regulation.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.10.14.618196

Protein (function)
Cas9: 
The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite

DiCre:
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 rapamycin‐binding protein (either FKBP12 or FRB, the rapamycin‐binding domain of the FKBP12‐rapamycin‐associated protein mTOR).
Rapamycin‐induced heterodimerization of the two components restores recombinase activity (rapamycin‐mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn60‐343, called Cre60) each fused to a different rapamycin‐binding protein (FKBP12 and FRB respectively) resulting in the reconstitution of Cre recombinase activity.

Phenotype
Transgenic PbCas-DiCre-GFP parasites complete their lifecycle normally, both in the vertebrate host and in the mosquito

Additional information

Other mutants