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. 2022 Jun 29;10(3):e0278221.
doi: 10.1128/spectrum.02782-21. Epub 2022 May 5.

A Leak-Free Inducible CRISPRi/a System for Gene Functional Studies in Plasmodium falciparum

Xiaoying Liang  1 , Rachasak Boonhok  1 , Faiza Amber Siddiqui  1 , Bo Xiao  2 , Xiaolian Li  1 , Junling Qin  1 , Hui Min  1 , Lubin Jiang  2 , Liwang Cui  1   3 , Jun Miao  1   3
Affiliations

A Leak-Free Inducible CRISPRi/a System for Gene Functional Studies in Plasmodium falciparum

Xiaoying Liang et al. Microbiol Spectr. .

Abstract

By fusing catalytically dead Cas9 (dCas9) to active domains of histone deacetylase (Sir2a) or acetyltransferase (GCN5), this CRISPR interference/activation (CRISPRi/a) system allows gene regulation at the transcriptional level without causing permanent changes in the parasite genome. However, the constitutive expression of dCas9 poses a challenge for studying essential genes, which may lead to adaptive changes in the parasite, masking the true phenotypes. Here, we developed a leak-free inducible CRISPRi/a system by integrating the DiCre/loxP regulon to allow the expression of dCas9-GCN5/-Sir2a upon transient induction with rapamycin, which allows convenient transcriptional regulation of a gene of interest by introducing a guide RNA targeting its transcription start region. Using eight genes that are either silent or expressed from low to high levels during asexual erythrocytic development, we evaluated the robustness and versatility of this system in the asexual parasites. For most genes analyzed, this inducible CRISPRi/a system led to 1.5- to 3-fold up-or downregulation of the target genes at the mRNA level. Alteration in the expression of PfK13 and PfMYST resulted in altered sensitivities to artemisinin. For autophagy-related protein 18, an essential gene related to artemisinin resistance, a >2-fold up- or downregulation was obtained by inducible CRISPRi/a, leading to growth retardation. For the master regulator of gametocytogenesis, PfAP2-G, a >10-fold increase of the PfAP2-G transcripts was obtained by CRISPRa, resulting in >4-fold higher gametocytemia in the induced parasites. Additionally, inducible CRISPRi/a could also regulate gene expression in gametocytes. This inducible epigenetic regulation system offers a fast way of studying gene functions in Plasmodium falciparum. IMPORTANCE Understanding the fundamental biology of malaria parasites through functional genetic/genomic studies is critical for identifying novel targets for antimalarial development. Conditional knockout/knockdown systems are required to study essential genes in the haploid blood stages of the parasite. In this study, we developed an inducible CRISPRi/a system via the integration of DiCre/loxP. We evaluated the robustness and versatility of this system by activating or repressing eight selected genes and achieved up- and downregulation of the targeted genes located in both the euchromatin and heterochromatin regions. This system offers the malaria research community another tool for functional genetic studies.

Keywords: DiCre; Plasmodium falciparum; dCas9; gene regulation; human malaria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Generation of inducible dCas9-GCN5/Sir2a systems using DiCre/loxP. (A) Schematic diagram illustrates the DiCre/loxP-inducible CRISPR/dCas9 system. GFP with stop codon and Hsp86 3′UTR flanked by loxP sequence was inserted before dCas9-GCN5/Sir2a. Rap is added to activate DiCre to remove sections between loxP sites, leading to the expression of dCas9-GCN5/Sir2a. FLAG, FLAG tag; D10A and H840A, two mutations in Cas9; NLS, nuclear localization signal; GS, glycine-serine protein domain linker. Two small black arrows in opposite directions show the locations of primers for PCR in panel C. The numbers between the arrows indicate the expected sizes of PCR products. (B) Live images and Western blots show the GFP and dCas9-fusion protein expression before (−Rap) and 48 h after (+Rap) DiCre was activated by the addition of Rap. (C) The efficiency of DiCre excision of loxPed GFP cassette was measured by PCR with specific primers shown in panel A. The gradual decrease and increase of unexcised and excised bands at the sizes 1,758 bp and 122 bp are shown in agarose gels, respectively. BF, bright field. (D) Three biological replicates were performed for the experiment in panel C to depict a bar graph showing the ratio of excised to unexcised bands over time elapsed after Rap treatment; pixel intensities of DNA bands were measured using ImageJ. (E) Western blots show the induction of dCas9-GCN5 and dCas9-Sir2a protein expression and decrease of GFP expression at ring (R), trophozoite (T), and schizont (S) stages after Rap treatment. Rap was added at 0 to 6 h postinvasion (hpi) for 2 h.
FIG 2
FIG 2
Repression and activation of selected genes by inducible dCas9 systems. (A) Transcriptional expression levels of eight selected genes during IDC based on the transcriptomic profile by RNA-seq. (B) Schematic diagrams show the location of TSSs and gRNAs in six selected genes. (C to E) The transcriptional changes of PfGCN5, PfCHC, and PfK13 in loxPed GFP-dCas9-GCN5 or Sir2a parasite lines after Rap treatment, respectively. (F) Left panel: RT-qPCR analysis of PfCRT transcript using 3 different sgRNAs (gRNA1 to 3) with dCas9-GCN5 and dCas9-Sir2a systems. Right panel: Western blot of PfCRT protein expression levels after activation and repression of PFCRT by using an anti-PfCRT antibody with anti-aldolase as a loading control. (G and H) The transcription levels of PfMYST and PfDNMT in the loxPed GFP-dCas9-GCN5 or Sir2a parasite lines after Rap treatment. Three replicates of RT-qPCR were performed for each gRNA and the paired t tests were conducted (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (I and J) Three replicates of recovery assays show the parasite growth after DHA treatment of parasite lines targeting PfK13 (I) and PfMYST (J) by dCas9-GCN5/Sir2a with respective gRNA1, compared to the parasite lines with gRNA but without Rap induction and the parasite lines without gRNA but with Rap induction.
FIG 3
FIG 3
Repression and activation of PfATG18 by inducible dCas9 system. (A) Schematic diagram shows the location of TSSs and gRNAs in the 5′ UTR of PfATG18. (B and D) The changes of PfATG18 transcriptions were analyzed by RT-qPCR in dCas9-Sir2a (B) and dCas9-GCN5 (D) parasite lines with gRNA1-3 after Rap treatment compared to the transcriptions without Rap treatment, respectively. Paired t tests were conducted (**, P < 0.01; ***, P < 0.001) from three replicates. (C and E) Parasite growth phenotype after repression and activation of PfATG18 using gRNA1-3 in dCas9-Sir2a (C) and dCas9-GCN5 (E) systems was investigated by measuring the parasitemia from three replicates, respectively. Compared to the growth rate in the parasites without Rap treatment (−Rap), parasites harboring gRNA1 in dCas9-Sir2a system grew significantly more slowly at day 5 posttreatment (+Rap) (***, P < 0.001, two-way ANOVA), whereas parasites harboring any gRNA (1/2/3) in dCas9-GCN5 system grew significantly more slowly at days 3 and 5 posttreatment (+Rap) (***, P < 0.001, two-way ANOVA).
FIG 4
FIG 4
Activation of AP2-G by the inducible dCas9-GCN5 system. (A) Schematic diagram shows the location of TSS and gRNAs in the 5′ UTR of AP2-G. The areas containing AP2-G/G5 binding motifs are labeled with three blue peaks. (B) A diagram illustrates the experimental workflow for the analysis of activation of AP2-G by the inducible system dCas9-GCN5. R, ring; T, trophozoite; S, schizont; Ga: gametocyte. (C) The changes of AP2-G transcription were analyzed by RT-qPCR in the parasites harboring inducible dCas9-GCN5 system and one of three gRNAs (gRNA1 to 3) compared to the transcription without Rap induction. Paired t tests were conducted (ns, not significant; ***, P < 0.001) from three replicates. (D) Three replicates of gametocytemia were counted 5 days after parasites harboring inducible dCas9-GCN5 system and gRNA3 were treated by Rap (+Rap) compared to the same parasites without Rap treatment (−Rap) (****, P < 0.0001, paired t test). (E) The two early gametocytes makers, pfgexp5 and pfs16, were analyzed by RT-qPCR in the parasites harboring the dCas9-GCN5 system and gRNA3 at 76 to 82 hpi when the gametocytogenesis started (stage 1) and asexual parasites were at the trophozoite stage. The paired t tests were conducted (**, P < 0.01, ***, P < 0.001) from three replicates.
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