Parasite culture, Synchronization, and Pellets collection. P. falciparum asexual stages (3D7 strain) were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. It was grown under 5% O2, and 5% CO2 in RPMI-1640 media supplemented with 5 g/L Albumax II (Life Technologies), 2 g/L sodium bicarbonate, 25 mM HEPES pH7.4 (pH adjusted with potassium hydroxide), 1 mM hypoxanthine and 50 mg/L gentamicin as previously described.
For synchronization, parasites were cultured to at least 10% parasitemia in T-75 flasks containing 50 ml medium at 1% hematocrit. Then it was moved from a flask to a 50 mL tube, centrifuged for 5 min at 500x g and removed supernatant. 15 mL 5% D-Sorbitol solution was added to the pellet and incubated at 37℃ for 10 min, centrifuged, and removed supernatant. The culture was synchronized with three rounds of sorbitol treatments. Then after the invasion at 8 h, 16 h, 24 h, 32 h, 40 h, 46/0 h collected and centrifuged culture solution, pellets were stored at -80℃ until to use.
Plasmid constructs and plasmodium transfection. Based on pUF1-Cas9 and pL6cs plasmids, kindly provided by Jose-Juan Lopez-Rubio, pUF1-BSD-Cas9 and pL6CS-hDHFR-hrp2 were constructed to disruption of the Pfhrp2 locus. The two plasmids and plasmodium transfection described as previous.
Briefly, pUF1-BSD-Cas9 expresses Cas9 nuclease and blasticidin S deaminase (BSD). The pL6CS-hDHFR-hrp2 plasmid, which offers donor DNAs and sgRNAs, targeting the Pfhrp2 gene (guidehrp2). The 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 construct of transitional- pL6CS-hDHFR-hrp2 was transformed into competent cells, and plasmids were extracted and checked with restriction enzyme and sequencing. After the correct transitional- pL6CS-hDHFR-hrp2 was obtained, this transitional plasmid was linearized with AvrII & XhoI. The sgRNAs of hrp2 were annealed and inserted into linearized transitional- pL6CS-hDHFR-hrp2 plasmids using the in-fusion kit. The construct was transformed into competent cells again and extracted. The final plasmid was confirmed by restriction enzyme digestion and DNA sequencing. The confirmed plasmid was isolated and used for electroporation to generate P. falciparum transgenic strains.
At the electroporation step, the two plasmids were 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 seconds. Drugs (final concentration is 2.5 mg/L blasticidin S and 250 mg/L G418) were added into complete medium post-transfection to kill those parasites without episomal pUF1-BSD-Cas9 and pL6CS-hDHFR-hrp2. All primers and sgRNA sequences used for constructing plasmids can be seen in Additional file 1.
Confirmation of transgene success via PCR. Twenty days after electroporation, live P. falciparum appeared, and genomic DNA was extracted from harvest parasite pellets using the Qiagen DNA extraction kit (QIAGEN, Valencia, California USA). A PCR was performed in 20 µl total volume 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 cultured P. falciparum parasite 3D7 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 sent for sequencing to confirm.
Western blotting analysis. Successfully transgenic parasites were cultured in flasks. For the analysis of Pfhrp2 expression, when parasitemia passed 5%, 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 was collected and added an appropriate amount of SDS-PAGE sample buffer, and denatured at 95℃ for 8 min, and resolved by electrophoresis in a 12.5% polyacrylamide gel (Life Technologies) and transferred onto a 0.2-um polyvinylidene difluoride (PVDF) membrane (Hybond LFP; GE Healthcare). HRP2 was specifically detected by using the Anti-Plasmodium falciparum monoclonal antibody [MPFG-55P] (HRP) (Abcam, Massachusetts, USA).
Southern Blot Analysis. The genomic DNA from transgenic parasites was isolated, as described above. For parasites, 5 µg of genomic DNA was digested overnight using the PstI or SacI restriction endonucleases (TaKaRa Bio companies). The DNAs were separated on a 1.0% agarose gel and transferred to Hybond™ –N+ membrane (GE Healthcare Amersham™) using the high salt capillary transfer method. Probes were PCR-amplified, cleaned, and labeled with DIG-dUTP using a PCR DIG Probe Synthesis Kit (Roche). The blots were hybridized with the labeled probes, washed, and exposed to film and detected by in a cassette.
Total RNA extraction. Total RNA was extracted using TRIzol® according to the manufacturer’s protocol. Briefly, different time collected pellets 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 room temperature, then centrifuged at 10,000 × g for 5 min at 4℃. The supernatant was added chloroform/isoamyl alcohol (24:1) with lysis reagents. After centrifuged at 10,000 × g for 10 min at 4℃, the supernatant was transferred into a new tube with an equal volume of isopropanol and kept at -20℃ for 1 h. After centrifuged at 13600 × g for 20 min at 4℃, the supernatant was precipitated by ethanol and dry for 3 min. The RNA pellet was dissolved with RNase-free water.
mRNA Library Construction. Oligo(dT)-attached magnetic beads were used to purified 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. Afterward, A-Tailing Mic and RNA Index Adapters were added by incubating to end repair. The cDNA fragments were amplified by PCR, and purified by Ampure XP Beads, then dissolved in EB solution. The double-stranded PCR products were heated to denature and circularized by the splint oligo sequence to get the final library. The single-strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB), which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray, and pair-end 100 bases reads were generated on BGISEQ500 platform (BGI-Shenzhen, China).
Sequencing Data Analysis. The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) removing reads containing sequencing adapter; (2) removing reads whose low-quality base ratio (base quality ≤ 5) is more than 20%; (3) removing reads whose unknown base (‘N’ base) ratio is more than 5%, the clean reads were stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, then expression level of the gene was calculated by RSEM (v1.2.12) and drawn by pheatmap (v1.0.8). Primally, differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05.