Synthetic RNA–protein modules integrated with native translation mechanisms to control gene expression in malaria parasites

Abstract

Synthetic posttranscriptional regulation of gene expression is important for understanding fundamental biology and programming new cellular processes in synthetic biology. Previous strategies for regulating translation in eukaryotes have focused on disrupting individual steps in translation, including initiation and mRNA cleavage. In emphasizing modularity and cross-organism functionality, these systems are designed to operate orthogonally to native control mechanisms. Here we introduce a broadly applicable strategy for robustly controlling protein translation by integrating synthetic translational control via a small-molecule-regulated RNA–protein module with native mechanisms that simultaneously regulate multiple facets of cellular RNA fate. We demonstrate that this strategy reduces ‘leakiness' to improve overall expression dynamic range, and can be implemented without sacrificing modularity and cross-organism functionality. We illustrate this in Saccharomyces cerevisae and the non-model human malarial parasite, Plasmodium falciparum. Given the limited functional genetics toolkit available for P. falciparum, we establish the utility of this strategy for defining essential genes.

The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

“…However, when aTc is removed, TetR‐DOZI binds the aptamer array and the RNA‐modifying ability of DOZI can target the mRNA for sequestration/degradation (Figure a; Ganesan et al . , ). Using CRISPR/Cas9, we successfully integrated a 3′ MYC tag/aptamer array at the PfTRiC‐θ locus, confirmed by Southern blotting (Figure Bi).…”

“…Introduction of the aptamer array in the 3′ UTR may alter the basal expression of the target protein (independently of TetR‐DOZI regulation; Ganesan et al . , ). This was not the case in the PfTRiC‐θ‐MYC parasites, as probing with the PfTRiC‐θ antibody revealed similar protein expression between the NF54 parent line and PfTRiC‐θ‐MYC parasites (Figure ai–iii; expression of PfTRiC‐θ‐MYC in clone B8 was 105 ± 11% of NF54, N = 2; mean ± range/2).…”

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes

“…We tagged the endogenous locus of Pf CLAMP with a FLAG epitope tag and ten tandem aptamer sequences, which bind the Tet Repressor protein (TetR) when transcribed (Ganesan et al, 2016). TetR is expressed as a fusion with the translational repressor (DOZI) in the same strain, which suppresses expression of the aptamer-tagged transcript unless anhydrotetracycline (aTc) is added to the media (Figure 6M).…”

“…P155 included an sgRNA targeting the 3′ end of the CLAMP locus, placed under the regulation of the T7 promoter in the final construct. The fragments were cloned by Gibson assembly into the plasmid containing the aptamers, and the Renilla luciferase-blasticidin deaminase fusion separated from the TetR-DOZI fusion by a T2A self-cleaving peptide, to generate p Pf CLAMP-cKD/TetR-DOZI (Ganesan et al, 2016). …”

“…Linearized p Pf CLAMP-cKD/TetR-DOZI was transfected into NF54 Cas9+T7 Polymerase . 50 μg of plasmid were used per 200 μl packed red blood cells (RBCs), adjusted to 50% haematocrit, and electroporated as previously described (Ganesan et al, 2016). Transfected parasites were selected with a combination of 2.5 μg/ml Blasticidin S and 2.5 nM WR99210 beginning 4 days after transfection.…”