A scaleable inducible knockout system for studying essential gene function in the malaria parasite

Abstract

The malaria parasite needs nearly half of its genes to propagate normally within red blood cells. Inducible ways to interfere with gene expression like the DiCre-lox system are necessary to study the function of these essential genes. However, existing DiCre-lox strategies are not well-suited to be deployed at scale to study several genes simultaneously. To overcome this, we have developed SHIFTiKO (frameshift-based trackable inducible knockout), a novel scaleable strategy that uses short, easy-to-construct, barcoded repair templates to insert loxP sites around short regions in target genes. Induced DiCre-mediated excision of the flanked region causes a frameshift mutation resulting in genetic ablation of gene function. Dual DNA barcodes inserted into each mutant enables verification of successful modification and induced excision at each locus and collective phenotyping of the mutants, not only across multiple replication cycles to assess growth fitness but also within a single cycle to identify specific phenotypic impairments. As a proof of concept, we have applied SHIFTiKO to screen the functions of malarial rhomboid proteases, successfully identifying their blood stage-specific essentiality. SHIFTiKO thus offers a powerful platform to conduct inducible phenotypic screens to study essential gene function at scale in the malaria parasite.

Graphical Abstract

Figure 1.

SHIFTiKO design. A short segment of length not divisible by three is replaced in the target gene with a recodonized sequence flanked by a pair of synthetic intron modules (boxit), each containing a loxP site (arrow head) and a unique 12 bp barcode. Gene modification is achieved by a Cas9-driven homology directed repair strategy in which double-stranded breaks are introduced in two gRNA target sites (shown near "Cas9") simultaneously. Treatment with rapamycin induces DiCre-mediated excision of the segment, resulting in a frameshift mutation to render the downstream segment of the gene non-functional. Binding sites for barcode-amplifying primers, barcode.F and barcode.R (half arrows) are placed in such a way that only the boxit- barcode is amplified from a modified shiftiko line and only the boxit+ barcode is amplified from the excised mutant.

Figure 2.

Induced disruption of SUB1 function using SHIFTiKO. (A) Diagnostic PCR amplification of the entire modified locus from sub1-shiftiko parasites at 24 h following RAP or mock treatment confirms efficient excision. Expected amplicon sizes are indicated. Long-read sequencing of the amplicons show precise excision of the floxed region containing the boxit- barcode whilst the boxit+ barcode is retained in the RAP-treated parasites. (B) Treatment with RAP results in ablation of growth in an uncloned transfectant population of sub1-shiftiko parasites. Data shown are averages from three biological replicates using different blood sources (shaded ribbon, ± SEM). (C) Light microscopic images of Giemsa-stained sub1-shiftiko parasites at 52 h following RAP and mock treatment at ring stages show that RAP-treated mutants are unable to undergo egress. Scale bar, 5 μm. (D) To amplify barcodes from RAP- and mock- treated sub1-shiftiko parasites, genomic DNA extracts were first digested with DpnI to selectively remove residual repair plasmids carried over from the transfectant cultures. Nested PCR results in 438 bp long amplicons that were sequenced on a ONT MinION device. Barcode counts show increase in boxit+ proportions in the RAP-treated population signifying efficient excision.

Figure 3.

Inducible phenotypic screening of Plasmodium rhomboids using SHIFTiKO. (A) Uniform floxing strategies to create shiftiko lines for the 13 target genes (for gene IDs, see Supplementary Figure S2). In each case, a short region upstream of the functional domain and containing two good scoring gRNA target sites was chosen to be floxed with boxit modules. Simultaneous tagging of the N-terminus with a 3xHA tag was successful for four of the eight rhomboids attempted. (B) SHIFTiKO workflow to assess fate of several inducible mutants during blood stage progression. Transfections and subsequent drug-selection for transfected parasites are carried out in arrayed format to produce individual shiftiko lines, that are then pooled together, expanded and synchronized to set up a culture of tightly synchronized ring stages. Following rapamycin (RAP) treatment, samples are collected at carefully chosen time points during four successive erythrocytic cycles to capture the informative changes in barcode abundances : Mock- (−RAP) and RAP-treated (+RAP) samples from 24 h post-treatment (T0) to inform on successful integration and excision; fully mature +RAP schizonts from cycle 0 (T0sz), newly formed rings (T1) and the remaining non-egressed schizonts (T0nsz) after letting +RAP schizonts from cycle 0 undergo egress and invade fresh RBCs, and +RAP schizonts from cycle 1 (T1sz) to inform on knockout phenotypes; and samples from cycle 2 and 3 (T2 and T3) in addition to T0 and T1 to inform on growth fitness. Barcodes are amplified from genomic DNA from the collected samples and sequenced on a ONT MinION device. (C) Rationale behind choosing sampling time points within cycle 0 and 1 to evaluate knockout phenotypes. Mutants with a defect in schizont (yellow) or trophozoite (purple) development are expected to have fewer DNA copies and therefore lower barcode abundances in T0sz and T1sz respectively, compared to non-defective mutants (green). Barcodes of mutants with an invasion defect (red) are expected to be depleted in newly formed ring fractions (T1) whilst those of mutants with an egress defect (orange) are selectively enriched in the non-egressed schizont fraction (T0nsz). (D) Proportions of boxit- and boxit+ barcodes in mock- (−RAP) and RAP-treated (+RAP) parasites (T0) show successful integration and excision in 9 out of 13 target genes. Data shown are averages from three replicate RAP treatments (error bars, ± SEM). (E) Relative growth fitness of knockout mutants (changes in boxit+ barcode proportions from T0, normalized to the non-essential p230p gene) reveal essential (red) and non-essential (blue) genes (shaded ribbon, 95% confidence interval of the ratio estimated using delta method). (F) Within-cycle changes in barcode proportions (log₂ ratios normalized to the non-essential p230p gene) reveal knockout phenotypes for the mutants (red, essential gene, blue, non-essential). Inferring the phenotypes from the first instance of aberrant change in barcode abundances during cycle progression, data reveals a schizont developmental defect (T0sz versus T0) in rom8 and piplc, an egress defect (T0nsz versus T0sz) in sub1 and an invasion defect (T1 versus T0sz) in rom4 mutants. Data shown are averages from three replicate experiments (error bars, ± SEM). (G) Light microscopic images of Giemsa-stained rom8-shiftiko schizonts allowed to fully mature in the presence of reversible egress inhibitor C2 following RAP (+RAP) and mock (−RAP) treatment at ring stages. ROM8-null parasites exhibit defective development producing abnormal schizonts, as confirmed and quantified using flow cytometry to measure parasite DNA content. Fluorescence intensity of the SYBR Green-stained RAP-treated population (red) was detectably lower than that of the mock-treated population (grey). Scale bar, 5 μm. (H) MSP1 (merozoite surface protein 1) localization in ROM8-null schizonts (+RAP) show evidence of segmentation to produce individual merozoites, albeit in significantly lower numbers compared to mock-treated controls (−RAP) (crossbar represents median; n= 12; Welch t-test with Bonferroni adjusted P-value). Scale bar, 5 μm. (I) Light microscopic images of Giemsa-stained RAP- and mock-treated rom4-shiftiko parasites after 4 h of invasion in static cultures. ROM4-null merozoites are unable to invade RBCs and form ring stages. Scale bar, 5 μm. (J) Fold change in parasitaemia after 4 h invasion of mock- (−RAP) and RAP-treated (+RAP) rom4-shiftiko parasites under shaking and static conditions. RAP-treated cultures show no increase in parasitaemia under static conditions but show a 5-fold increase in shaking cultures, albeit still lower than mock-treated controls (crossbar represents median, three replicate RAP treatments with different blood sources; individual points represent each replicate). Fluorescence intensity of SYBR Green-stained parasite populations pre- (0 h) and post-invasion (4 h) confirm that ROM4-null mutants undergo normal egress (causing the disappearance of the schizont peak) and that the increase in parasitaemia in shaking cultures is due to formation of ring stages.