Generating conditional gene knockouts in Plasmodium - a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9

Abstract

Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. Here we employ CRISPR/Cas9 to facilitate use of the dimerisable Cre-recombinase (DiCre) that is frequently used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin controlled manner. We describe novel CRISPR/Cas9 transfection plasmids and approaches for the speedy, stable and marker-free introduction of transgenes encoding the DiCre recombinase into genomic loci dispensable for blood stage development. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. Furthermore, the newly developed 3D7-derived parasite lines, constitutively and stably expressing DiCre, generated using this toolkit will prove useful for the analysis of gene products. Lastly, we introduce an improved treatment protocol that uses a lower rapamycin concentration and shorter treatment times, leading to loxP-guided recombination with close to 100% efficiency within the same replication cycle.

Figure 1

Optimisation of rapamycin-induced recombination. (a) Parasites containing a modified PFA0210c were used to test DiCre-mediated recombination efficiency. For this a recodonised version of part of the ORF of PFA0210c was flanked by a loxPint sequence upstream and a loxP sequence downstream located within the endogenous locus on chromosome 1 in the 1G5DC parasite line. (b) The efficiency of excision was tested whilst altering the concentration and length of exposure to rapamycin (rapa). Either DMSO alone (0 nM) or increasing concentrations (in nM) of rapamycin were added to transgenic PFA0210c-loxP parasites as indicated for 4, 2, 1 h or 30 min. The degree of recombination was determined by PCR using primers 13 and 14 depicted as arrows in panel a). A lack of recombination and no excision resulted in a PCR product of 1580 bp (u) whereas successful excision resulted in a 562 bp product (e). The sizes of DNA markers (in kb) are depicted on the left. (c) The timing of excision was determined following addition of either 100 nM rapamycin for 4 h (100) or 10 nM rapamycin for 30 min (10) at very early ring stages. Parasites were harvested 2, 4, 8, 20, 30 or 44 h after the onset of treatment. Genomic DNA was extracted from saponin-treated parasite pellets and excision was determined by PCR as described in panel b. (d) Graph depicting the degree of excision (in %) over time elapsed after start of rapamycin treatment. For this graph, the data in panel c) was semi-quantitatively analysed. Pixel intensities of stained DNA bands were measured using ImageJ and the ratio of excised vs. unexcised was plotted against time after rapamycin addition, comparing two treatment methods (100 nM rapamycin for 4 h and 10 nM rapamycin for 30 min).

Figure 2

Toolkit plasmids for efficient integration of DiCre recombinase into p230p or pfs47 loci of the P. falciparum genome. The CRISPR-Cas9 plasmid used here, pDC2-Cas9-hDHFRyFCU (derived from pDC2-Cas9-U6-hdhfr ) carries the SpCas9 nuclease gene and a single guide RNA cassette driven by a short U6 promoter and the hdhfr-yfcu fusion gene for selection. The rescue plasmids (pBSp230pDiCre and pBSPfs47DiCre) contain the genes for the split Cre-recombinase fused to FRB and FKBP12 respectively, flanked by regions of homology from the p230p or the pfs47 locus of P. falciparum. More detailed vector maps are available in Supplementary Fig. S2.

Figure 3

Successful generation of P. falciparum parasite lines expressing DiCre recombinase using CRISPR-Cas9. (a) Schematic of wild type p230p ORF and modified ORF containing the DiCre recombinase expression cassettes following transfection of 3D7 with pBSp230pDiCre and pDC120 (pDC2-hDHFR-yFCU plasmid carrying the guide sequence). Homology regions (HRs) used for targeting were 500 bp (HR1) and 644 bp (HR2) in size. (b) Schematic of wild type pfs47 locus and modified, DiCre-expressing pfs47 locus. The blue box depicts the pfs47 ORF, and hashed boxes the DNA regions used for homologous repair. Transfection of 3D7 with pBSPfs47DiCre and pDC287 (the plasmid carrying the guide sequence for Pfs47 targeting) resulted in the disruption of the pfs47 ORF. (c) PCR screen on wild type and transgenic parasite clones (II-3 and Pfs47-13) showing successful integration of the DiCre cassette into the targeted loci. Primer pairs 1/2 and 3/4 amplify fragments of p230p in wild type parasites only with expected sizes of 772 bp and 771 bp respectively. Primer pairs 1/5 and 4/6 amplify DNA only after successful integration into p230p with expected sizes of 947 bp and 1229 bp respectively. Primer pairs 7/8 and 9/10 amplify 1042 bp and 1114 bp fragments from the pfs47 wild type locus only; whereas primer pairs 5/7 and 6/10 only amplify the expected 1257 bp and 1555 bp bands after successful integration. Primers used for amplification are shown on the top of each panel, as well as in the schematics in a) and b). DNA marker sizes are indicated on the left in kb.

Figure 4

The new transgenic parasite lines excise floxed DNA sequences efficiently. (a) Schematic of reporter plasmid pHH3-SP-loxPint-GFP-loxP. The gfp ORF is flanked by a loxPint site upstream and a loxP site downstream. Transcription is driven by 1258 bp of msp3 5′UTR sequence. SP, 110 bp of signal peptide-encoding sequence from msp3; bsd, blasticidin S deaminase driven by hsp86 promoter. 3D7, and the DiCre-expressing parasite lines 1G5DC, II-3 and Pfs47-13 were transfected with the reporter plasmid and selected using 2.5 µg/ml blasticidin. (b) PCR amplification of the GFP cassette using primers 11 and 12 showing successful recombination after rapamycin treatment. The expected product size is 1471 bp before excision and 670 bp following excision. Treatment conditions were either 0.05% DMSO (0), 100 nM rapamycin for 4 h (100) or 10 nM rapamycin for 1 h (10) as indicated on the top of each lane. The parasite lines treated are indicated at the bottom of the panel. DNA marker sizes are shown on the left. (c) Immunoblots showing the lack of a 27 kDa GFP band after rapamycin treatment. The four parasite lines transfected with the reporter plasmid were treated with DMSO or rapamycin as indicated on the top of each panel and described in section b). Treatment occurred at early ring stage and schizonts were harvested by Percoll gradient 38–40 h later before separation on SDS-PAGE and immunoblotting. Anti-GFP and anti-MSP2 antibodies were used as indicated on the right of the panels.

Figure 5

The new transgenic parasite lines expressing DiCre recombinase excise floxed DNA sequences with >99% efficiency using rapamycin at lower concentrations and short treatment times. (a) Live GFP fluorescence assay of wild type and DiCre transgenic parasite lines transfected with the reporter plasmid pHH3-SP-loxPint-GFP-loxP and treated at the early ring stage with DMSO or rapamycin as indicated on top of the panels. Images were taken 40 h later. Size bars equal 10 μm. (b) Excision efficiency determined by flow cytometry. The four parasite lines transfected with the reporter plasmid were treated with DMSO or rapamycin as described above and the number of GFP positive schizonts was counted 38–40 h after treatment. Data correspond to the mean and standard deviation (s.d.) calculated from three independent experiments counting 100,000 cells each time.