CRISPR-Cas9 genome editing of Plasmodium knowlesi

Mohring, Franziska; Hart, Melissa N.; Patel, Avnish; Baker, David A.; Moon, Robert W.

doi:10.21769/bioprotoc.3522

Cited by 18 publications

(11 citation statements)

References 11 publications

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“…Mature schizont cultures at >6% parasitaemia were synchronised using 55% Nycodenz (Alere technologies AS) [ 33 ]. Cultures were centrifuged and media removed to leave 2ml of media and blood, which was layered gently on top of 5 mL of prewarmed 55% Nycodenz, then centrifuged at 1300g for 5 mins.…”

Section: Methodsmentioning

confidence: 99%

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

McDonald

Merrick

2022

PLoS Pathog

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Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This involves the asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite reproduction is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear multiplications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active DNA replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their DNA replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

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Section: Methodsmentioning

confidence: 99%

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

McDonald

Merrick

2022

PLoS Pathog

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“…The CRISPR-Cas9 genome editing of P. knowlesi has been established [ 193 ], which will definitely drive the rapid and effective creation of transgenic parasite lines with precise gene editing, knock-out, or addition of tags to facilitate the downstream functional analysis of the parasite gene candidates. Moreover, this method can be used in combination with the conditional knockout system to generate parasite lines that are stable for inducible gene deletions to study the essential genes.…”

Section: Introductionmentioning

confidence: 99%

Plasmodium knowlesi: the game changer for malaria eradication

Lee

Cheong

Amir

et al. 2022

Malar J

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Plasmodium knowlesi is a zoonotic malaria parasite that has gained increasing medical interest over the past two decades. This zoonotic parasitic infection is prevalent in Southeast Asia and causes many cases with fulminant pathology. Despite several biogeographical restrictions that limit its distribution, knowlesi malaria cases have been reported in different parts of the world due to travelling and tourism activities. Here, breakthroughs and key information generated from recent (over the past five years, but not limited to) studies conducted on P. knowlesi were reviewed, and the knowledge gap in various research aspects that need to be filled was discussed. Besides, challenges and strategies required to control and eradicate human malaria with this emerging and potentially fatal zoonosis were described.

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“…The methods developed here will be used to generate P. knowlesi genomes from patient isolates with matched metadata for parasite genome-wide disease association analyses. Experimental Plasmodium knowlesi is particularly receptive to genome editing, facilitating allele-specific phenotyping ( Mohring et al, 2020 ). Parasites edited with clinically relevant disease-associated alleles can be taken forward and characterized in vitro and in vivo for cause and effect.…”

Section: Discussionmentioning

confidence: 99%

De Novo Assembly of Plasmodium knowlesi Genomes From Clinical Samples Explains the Counterintuitive Intrachromosomal Organization of Variant SICAvar and kir Multiple Gene Family Members

et al. 2022

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Plasmodium knowlesi, a malaria parasite of Old World macaque monkeys, is used extensively to model Plasmodium biology. Recently, P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes uncomplicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum. As such, P. knowlesi presents a unique opportunity to develop experimental translational model systems for malaria pathophysiology informed by clinical data from same-species human infections. Experimental lines of P. knowlesi represent well-characterized genetically stable parasites, and to maximize their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization. The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence and de novo assemble P. knowlesi genomes from frozen clinical samples. The sequencing platform and assembly pipelines were designed to facilitate capturing data and describing, for the first time, P. knowlesi schizont-infected cell agglutination (SICA) var and Knowlesi-Interspersed Repeats (kir) multiple gene families in parasites acquired from nature. The SICAvar gene family members code for antigenically variant proteins analogous to the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) multiple var gene family. Evidence presented here suggests that the SICAvar family members have arisen through a process of gene duplication, selection pressure, and variation. Highly evolving genes including PfEMP1family members tend to be restricted to relatively unstable sub-telomeric regions that drive change with core genes protected in genetically stable intrachromosomal locations. The comparable SICAvar and kir gene family members are counter-intuitively located across chromosomes. Here, we demonstrate that, in contrast to conserved core genes, SICAvar and kir genes occupy otherwise gene-sparse chromosomal locations that accommodate rapid evolution and change. The novel methods presented here offer the malaria research community not only new tools to generate comprehensive genome sequence data from small clinical samples but also new insight into the complexity of clinically important real-world parasites.

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CRISPR-Cas9 genome editing of Plasmodium knowlesi

Cited by 18 publications

References 11 publications

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Plasmodium knowlesi: the game changer for malaria eradication

De Novo Assembly of Plasmodium knowlesi Genomes From Clinical Samples Explains the Counterintuitive Intrachromosomal Organization of Variant SICAvar and kir Multiple Gene Family Members

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