Efficient generation of mNeonGreen <i>Plasmodium falciparum</i> reporter lines enables quantitative fitness analysis

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation

A Roadmap for the Development of Ivermectin as a Complementary Malaria Vector Control Tool.

In the context of stalling progress against malaria, resistance of mosquitoes to insecticides, and residual transmission, mass drug administration (MDA) of ivermectin, an endectocide used for neglected tropical diseases (NTDs), has emerged as a promising complementary vector control method. Ivermectin reduces the life span of Anopheles mosquitoes that feed on treated humans and/or livestock, potentially decreasing malaria parasite transmission when administered at the community level. Following the publication by WHO of the preferred product characteristics for endectocides as vector control tools, this roadmap provides a comprehensive view of processes needed to make ivermectin available as a vector control tool by 2024 with a completely novel mechanism of action. The roadmap covers various aspects, which include 1) the definition of optimal dosage/regimens for ivermectin MDA in both humans and livestock, 2) the risk of resistance to the drug and environmental impact, 3) ethical issues, 4) political and community engagement, 5) translation of evidence into policy, and 6) operational aspects of large-scale deployment of the drug, all in the context of a drug given as a prevention tool acting at the community level. The roadmap reflects the insights of a multidisciplinary group of global health experts who worked together to elucidate the path to inclusion of ivermectin in the toolbox against malaria, to address residual transmission, counteract insecticide resistance, and contribute to the end of this deadly disease

New approaches to antimalarial target deconvolution and measuring fitness effects of Plasmodium falciparum mutations implicated in drug resistance.

Plasmodium falciparum remains one of the greatest global public health challenges. Current malaria control efforts face the persistent challenge of drug resistance and, to date, the malaria parasite has been able to develop resistance to almost every drug with which it has been challenged. One strategy to tackle the problem of drug resistance is to systematically identify new antimalarials to which the parasite has not yet developed resistance. Many novel compounds with antimalarial activity are in development. However, in most cases, the targets and the mechanisms of action of these compounds are unknown. Understanding which compounds are targeting new pathways for which no resistance exists is critical to prioritising novel drug candidates. Understanding drug resistance in more depth has the added advantage that targeting pathways for which resistance comes at a high fitness cost could prolong drug efficacy in the field. There may also be cases where drug resistance mutations confer hypersensitivity to new antimalarials compounds. Identifying cases where this occurs would allow us to develop better combination therapies. Currently, it is only practical to test the effectiveness of new antimalarials against a handful drug- resistant mutants at a time. To develop a new platform to address this problem, I set out to use BarSeq technology and CRISPR-Cas9 editing to create a pool of barcoded drug resistant mutants that covered a diverse representation of the currently known malaria resistome. These mutants would include three main groups: 1. Lines with clinical significance: mutations that confer resistance to antimalarial compounds currently being used in the field. 2. Lines with significance to compounds in the Medicines for Malaria Venture (MMV) pipeline: mutations that confer resistance to compounds currently in the drug development pipeline. 3. Putative future drug targets: resistance mutations generated to “toolbox” compounds not currently in the drug development pipeline. By utilising CRISPR-Cas9 genome editing technology, barcode cassettes were inserted into the genome of drug-resistant lines. These lines were then combined into one master pool that would allow all lines to be interrogated in parallel. Amplicon sequencing was then used to compare the relative growth of each mutant over time in the presence and absence of query compounds. This would allow multiplex competition assays to identify the targets or mechanisms of resistance to novel antimalarials. The ultimate aim is to develop a comprehensive cross-resistance screening platform for rapid compound profiling. The initial part of my work focused on improving the efficiency of the CRISPR-based barcode insertion reaction, targeting the Pfrh3 locus, that had previously been developed in the lab. When I attempted this approach on a larger scale, it was found that editing was too inefficient with only 5% of transfections being successfully barcoded. Several different editing strategies to increase barcoding efficiency were explored. In the end, a positive and negative selection approach was used to enrich for edited parasites and to insert barcodes into a new nonessential locus, Pfpare. This increased the percentage of successfully barcoded transfections to over 50% with all parasites being edited in these successful transfections. This increase in efficiency has allowed me to insert 53 barcodes into 47 different drug-resistant lines, covering the lines of clinical significance, lines with significance to compounds in the MMV pipeline, and the majority of the putative future antimalarial targets Drug resistant mutants were pooled together and grown in competition in the absence of the drug to measure fitness costs of resistance. The pool was tested with well-characterised antimalarials GNF179 and NITD609 to optimise screening parameters including selection concentration and time of harvest. The pool was then validated using a panel of blinded compounds with different mechanisms of action supplied by the MMV. Testing of the pool revealed that it could be used to select out specific mutant lines that had been shown to confer resistance to these compounds. Antimalarial compounds with novel mechanisms of action were successfully identified using the BarSeq approach because of their profile of total killing of the pool, indicating no relevant resistant lines were present. However, to provide insight into their mechanism of action, in vitro evolution experiments or other compound profiling experiments would still need to take place. The rate at which resistant mutations can be generated is limited by the mutation rate of the parasites in the selection. The use of so called hyper-mutator lines that have lost the ability to proofread during transcription has been established as a way to increase mutation rate and therefore the rate at which resistance is able to develop in yeast and bacteria. Recently our lab generated a Plasmodium falciparum line with this phenotype. Drug selection assays have shown that it is capable of developing resistance at a lower inoculum of parasites than its wildtype parent line. In this thesis, I investigated whether introducing a second source of mutation, in this case UV radiation, could accelerate this process even further. I established a protocol for treating parasites with UV radiation, then challenged UV and non-UV treated hyper-mutator and wildtype parasites with the antimalarial compound NITD609. The hyper- mutator line once again proved more able to develop resistance at lower concentrations than the wildtype parental line. The UV treated parasites returned more quickly after selection, however with only a modest improvement in time after selection. Nonetheless, this approach may be beneficial for compounds for which resistance is more challenging to generate

DataSheet_1_Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis.pdf

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.</p

Table_1_Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis.xlsx

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.</p

Genome-wide DNA methylation analysis of patients with imprinting disorders identifies differentially methylated regions associated with novel candidate imprinted genes

BACKGROUND: Genomic imprinting is allelic restriction of gene expression potential depending on parent of origin, maintained by epigenetic mechanisms including parent of origin-specific DNA methylation. Among approximately 70 known imprinted genes are some causing disorders affecting growth, metabolism and cancer predisposition. Some imprinting disorder patients have hypomethylation of several imprinted loci (HIL) throughout the genome and may have atypically severe clinical features. Here we used array analysis in HIL patients to define patterns of aberrant methylation throughout the genome. DESIGN: We developed a novel informatic pipeline capable of small sample number analysis, and profiled 10 HIL patients with two clinical presentations (Beckwith–Wiedemann syndrome and neonatal diabetes) using the Illumina Infinium Human Methylation450 BeadChip array to identify candidate imprinted regions. We used robust statistical criteria to quantify DNA methylation. RESULTS: We detected hypomethylation at known imprinted loci, and 25 further candidate imprinted regions (nine shared between patient groups) including one in the Down syndrome critical region (WRB) and another previously associated with bipolar disorder (PPIEL). Targeted analysis of three candidate regions (NHP2L1, WRB and PPIEL) showed allelic expression, methylation patterns consistent with allelic maternal methylation and frequent hypomethylation among an additional cohort of HIL patients, including six with Silver–Russell syndrome presentations and one with pseudohypoparathyroidism 1B. CONCLUSIONS: This study identified novel candidate imprinted genes, revealed remarkable epigenetic convergence among clinically divergent patients, and highlights the potential of epigenomic profiling to expand our understanding of the normal methylome and its disruption in human disease