Parasite culture, synchronization, and pellet collection
P. falciparum (3D7 strain) cells at the asexual stage were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. The parasite was grown under 5% O2 and 5% CO2 in RPMI-1640 medium supplemented with 5 g/L Albumax II (Life Technologies), 2 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4, adjusted with potassium hydroxide), 1 mM hypoxanthine, and 50 mg/L gentamicin as previously described [18].
For synchronization, parasite cells were cultured to at least 10% parasitaemia in T-75 flasks containing 50 ml of medium at 1% haematocrit. Then, the cells were moved from the flask to a 50-mL tube and centrifuged for 5 min at 500×g, and the supernatant was removed. Fifteen millilitres of 5% D-sorbitol solution was added to the pellet, followed by incubation at 37°C for 10 min, centrifugation, and removal of the supernatant. The culture was synchronized with three rounds of sorbitol treatment. Then, the invasion culture solution was collected at 8 h, 16 h, 24 h, 32 h, 40 h, and 46/0 h; 3 replicates were collected at each time point. A total of thirty-six samples were collected and centrifuged, and pellets were stored at -80°C until use.
Plasmid constructs and Plasmodium transfection
Based on the pUF1-Cas9 and pL6CS plasmids kindly provided by Jose-Juan Lopez-Rubio, pUF1-BSD-Cas9 and pL6CS-hDHFR-hrp2 were constructed to disrupt the Pfhrp2 locus (Fig. 1). Construction of the two plasmids and Plasmodium transfection were performed as described previously [19].
Briefly, pUF1-BSD-Cas9 expresses Cas9 nuclease and blasticidin S deaminase (BSD). The pL6CS-hDHFR-hrp2 plasmid expresses donor DNAs and sgRNAs targeting the Pfhrp2 gene (guidehrp2), and its left and right homologous arms were amplified separately by PCR from the genomic DNA of P. falciparum 3D7 (primers P1/P2 for the left arm and P3/P4 for right arm). The left homologous arm of hrp2 was ligated between the ASCI and AfLII sites of the pL6CS plasmid. The right homologous arm was ligated between the HindIII and EcoRI sites of the plasmid. The right homologous arm also used as a stop codon for the gene encoding hDHFR. The annealing reaction solution consisted of 1 μL of forward oligo (100 µM), 1 μL of reverse oligo (100 µM), and 23 μL of ddH2O. The annealing conditions for the sgRNA were as follows: 2 cycles of 30 s at 94°C and 30 s at 72°C, followed by 30 s at 60°C, 30 s at 25°C, and 30 s at 16°C. Then, the annealed sgRNA was ligated into the pL6CS plasmid.
The transitional construct pL6CS-hDHFR-hrp2 was transformed into competent cells, and plasmids were extracted and checked with restriction enzyme digestion and sequencing. After the correct transitionalpL6CS-hDHFR-hrp2 construct was obtained, this transitional plasmid was linearized with AvrII and XhoI. The sgRNAs of hrp2 were annealed and inserted into linearized transitional-pL6CS-hDHFR-hrp2 plasmid using the In-Fusion kit. The construct was transformed into competent cells again and extracted. The final plasmid was verified by restriction enzyme digestion and DNA sequencing. The confirmed plasmid was isolated and used for electroporation to generate transgenic P. falciparum strains.
Electroporation of the two plasmids was carried out by the spontaneous uptake method using ~50 μg of maxi-prepped plasmid DNA and 8 square wave electroporation pulses of 356 V for 1 ms each, separated by 0.1 s. Drugs (final concentration of 2.5 mg/L for blasticidin S and 5 nmol/L for WR99210) were added into the complete medium post-transfection to kill the parasites that lacked episomal pUF1-BSD-Cas9 and pL6CS-hDHFR-hrp2. The location of sgRNA in the hrp2 and gene disruption schematic are listed in Additional file 1: Figure S1. All primer and sgRNA sequences used for constructing plasmids are listed in Additional file 2: Table S1 and Additional file 3: Table S2.
Confirmation of successful transgene introduction via PCR
Twenty days after electroporation, live P. falciparum cells appeared, and genomic DNA was extracted from harvested parasite pellets using the Qiagen DNA Extraction Kit (Qiagen, Valencia, California USA). PCR was performed in a total volume of 20 μL, consisting of 10× buffer with 15 nM MgCl2, 200 μM dNTPs, 15 μM forward and reverse primers (P1/P2, as indicated in Fig. 2), 0.69 units of Taq DNA polymerase, and 2 μL of DNA template. An in vitro P. falciparum 3D7 culture was used as a positive control for Pfhrp2 gene amplification experiments. All PCR products were separated and visualized on agarose gels, and products with the expected size were submitted for sequencing for further confirmation.
Southern blotting analysis
Genomic DNA was isolated from transgenic parasites as described above. In total, 5 µg of the parasite genomic DNA was digested overnight using PstI or SacI restriction endonuclease (TaKaRa Bio). The DNA products were separated on a 1.0% agarose gel and transferred to a HybondTM –N+ membrane (GE Healthcare AmershamTM) using the high-salt capillary transfer method. Probes were PCR amplified, cleaned, and labelled with DIG-dUTP using the PCR DIG Probe Synthesis Kit (Roche). The blots were hybridized with the labelled probes, washed, and exposed to film and visualized in a cassette. All primer used for southern blotting analysis are listed in Additional file 4: Table S3.
Western blotting analysis
Successfully generated transgenic parasites were cultured in flasks. For the analysis of Pfhrp2 expression, when parasitaemia exceeded 5%, infected red blood cells (iRBCs) were collected and incubated with 0.15% saponin lysis solution on ice for 7 min. After centrifugation at 500×g for 5 min at room temperature (RT), the supernatants were collected and added to an appropriate amount of SDS-PAGE sample buffer, denatured at 95°C for 8 min, resolved by electrophoresis in a 12.5% polyacrylamide gel (Life Technologies) and transferred onto a 0.2-µm polyvinylidene difluoride (PVDF) membrane (Hybond LFP; GE Healthcare). HRP2 was specifically detected by using an anti-Plasmodium falciparum monoclonal antibody [MPFG-55P] (horseradish peroxidaseP) (Abcam, Massachusetts, USA).
Total RNA extraction
Total RNA was extracted using TRIzol according to the manufacturer’s protocol. Briefly, pellets collected at different times from transgenic parasites were ground into powder by liquid nitrogen and transferred into a new tube with TRIzol reagent. The mix was shaken and kept for 5 min at RT and then centrifuged at 10,000×g for 5 min at 4°C. Chloroform/isoamyl alcohol (24:1) was added to the supernatant with lysis reagents. After centrifugation at 10,000×g for 10 min at 4°C, the supernatant was transferred into a new tube with an equal volume of isopropanol and kept at -20°C for 1 h. After centrifugation at 13,600×g for 20 min at 4°C, the supernatant was precipitated by ethanol and dried for 3 min. The RNA pellet was dissolved with RNase-free water.
mRNA library construction and sequencing
Oligo (dT)-attached magnetic beads were used to purify mRNA from parasite pellets. Purified mRNA was fragmented into small pieces with buffer at the appropriate temperature. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Then, A-tailing mix and RNA index adapters were added via incubation for end repair. The cDNA fragments were amplified by PCR, purified by Ampure XP Beads, and then dissolved in EB solution. The double-stranded PCR products were heated to denature and circularized by the splint oligo sequence to obtain the final library. The single-strand circular DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoballs (DNBs), which had more than 300 copies of one molecule. DNBs were loaded into the patterned nanoarray, and paired-end 100-base reads were generated on the BGISEQ500 platform (BGI-Shenzhen, China).
RNA-Seq data analysis
The sequencing data were filtered with SOAPnuke (v1.5.2) [20] by (1) removing reads containing sequencing adapters; (2) removing reads with low-quality base (base quality ≤5) ratios more than 20%; (3) removing reads with unknown base (‘N’ base) ratios more than 5%. The clean reads were stored in FASTQ format. The clean reads were mapped to the reference P. falciparum 3D7 genome (assembly GCA_000002765) using HISAT2 (v2.0.4)[21]. Bowtie2 (v2.2.5) [22] was applied to align the clean reads to the reference coding gene set, and then, the gene expression levels were calculated by RSEM (v1.2.12)[23] and normalized to fragments per kilobase of transcript per million mapped reads (FPKM) values. Functional annotation of genes was achieved by mapping genes to the Gene Ontology (GO, http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) databases using BLAST software (V2.2.23). GO annotation was performed by Blast2GO (v 2.5.0) with NR annotations. DESeq2 (v1.4.5)[24] was used to detect differentially expressed genes (DEGs), and DEGs with fold change > 2 or < -2 and adjusted P value ≤ 0.001 were considered to be significant DEGs. GO enrichment analysis was performed using Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution), a function of R. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05).