Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis

Tůmová, Pavla; Dluhošová, Jana; Weisz, Filip; Nohýnková, Eva

doi:10.1016/j.ijpara.2019.01.003

Cited by 12 publications

(22 citation statements)

References 26 publications

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“…By use of fluorescence in-situ hybridization, we were able to detect individual loci of GL50803_16374 in nuclear spreads of Giardia wild-type strain. We confirmed previously described observations that this gene is completely absent from one of the two nuclei (Figure 4A) (Tůmová et al, 2019). The observed hybridization frequency in nuclei was 50.66 % (n=454 nuclei).…”

Section: Resultssupporting

confidence: 92%

“…intestinalis is considered tetraploid (Morrison et al, 2007;Xu et al, 2020), resulting in the need of knocking-out four copies of the gene to establish full knock-out. Thus, we first tested the system on the GL50803_16374 gene (mem), one of the few known genes present exclusively in one of Giardia's two nuclei (Tůmová et al, 2019). This would allow us the need to knock-out 'only' two copies in the same nucleus rather than four copies in two nuclei.…”

Section: Resultsmentioning

confidence: 99%

“…Giardia intestinalis has been eluding the use of this powerful technique so far. This issue may be addressed to the facts that (i), with few exceptions (Tůmová et al, 2016(Tůmová et al, , 2019, its genome harbors all the genes in four copies (Morrison et al, 2007;Xu et al, 2020) and (ii) its sex called diplomixis is limited exclusively to encystation (Poxleitner et al, 2008). Until recently (McInally et al, 2019), not even expression of full-length Cas9 nuclease in Giardia nuclei has been achieved (Ebneter et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

“…Giardia intestinalis is a binucleated protozoan parasite colonizing small intestine of various mammals (Einarsson, Ma'ayeh, et al, 2016). With exceptions (Tůmová et al, 2016(Tůmová et al, , 2019, Giardia is considered tatraploid as both its nuclei harbor two copies of each gene (Morrison et al, 2007;Xu et al, 2020). Although this actual tetraploidy and lack of defined sexual cycle (Poxleitner et al, 2008) proved Giardia difficult to genetically manipulate, there has been handful of studies showing its possibility.…”

Section: Introductionmentioning

confidence: 99%

“…We used in-vitro developed Cas9-guideRNA ribonucleoprotein complex to edit Giardia genome. Furthermore, by introducing 'self-propagating' CRISPR/Cas system, we knocked-out mem gene (GL50803_16374) that is harbored only by one nucleus (Tůmová et al, 2019). We also used the same strategy to completely knock-out cwp1 and mlf1, genes that were used as a targets for genomic manipulation previously (Ebneter et al, 2016;Lin et al, 2019) without achieving full knock-outs.…”

Section: Introductionmentioning

confidence: 99%

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Cas9-mediated gene disruption in tetraploidGiardia intestinalis

Horáčková

Voleman

Hagen

et al. 2021

Preprint

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CRISPR/Cas9 system is an extremely powerful technique that is extensively used for different genome modifications in various organisms including parasitic protists. Giardia intestinalis, a protozoan parasite infecting large number of people around the world each year, has been eluding the use of CRISPR/Cas9 technique so far which may be caused by its rather complicated genome containing four copies of each gene in its two nuclei. Apart from only single exception (Ebneter et al., 2016), without the use of CRISPR/Cas9 technology in its full potential, researchers in the field have not been able to establish knock-out cell lines to study the functional aspect of Giardia genes. In this work, we show the ability of in-vitro developed CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. Moreover, we used self-propagating CRISPR/Cas9 system to establish full knock out cell lines for mem, cwp1 and mlf1 genes. We also show that the system function even for essential genes, as we knocked-down tom40, lowering the amount of Tom40 protein by more than 90%. Further, we tested the length of homologous arms needed for successful integration of homology recombination cassette used for genome editing. Taken together, our work introduces CRISPR/Cas9 to Giardia for routine use in the lab, further extending the catalogue of molecular tolls available for genetic manipulation of the protist and allowing researchers to study the function of Giardia genes properly for the first time.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cas9-mediated gene disruption in tetraploidGiardia intestinalis

Horáčková

Voleman

Hagen

et al. 2021

Preprint

Self Cite

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Foraminifera as a model of the extensive variability in genome dynamics among eukaryotes

et al. 2022

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Knowledge of eukaryotic life cycles and associated genome dynamics stems largely from research on animals, plants, and a small number of "model" (i.e., easily cultivable) lineages. This skewed sampling results in an underappreciation of the variability among the many microeukaryotic lineages, which represent the bulk of eukaryotic biodiversity. The range of complex nuclear transformations that exists within lineages of microbial eukaryotes challenges the textbook understanding of genome and nuclear cycles. Here, we look in-depth at Foraminifera, an ancient (∼600 million-year-old) lineage widely studied as proxies in paleoceanography and environmental biomonitoring.We demonstrate that Foraminifera challenge the "rules" of life cycles developed largely from studies of plants and animals. To this end, we synthesize data on foraminiferal life cycles, focusing on extensive endoreplication within individuals (i.e., single cells), the unusual nuclear process called Zerfall, and the separation of germline and somatic function into distinct nuclei (i.e., heterokaryosis). These processes highlight complexities within lineages and expand our understanding of the dynamics of eukaryotic genomes.

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Giardiasis from a One Health Perspective

Lalle

Cacciò

2022

Zoonoses: Infections Affecting Humans and Animals

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Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis

Cited by 12 publications

References 26 publications

Cas9-mediated gene disruption in tetraploidGiardia intestinalis

Cas9-mediated gene disruption in tetraploidGiardia intestinalis

Foraminifera as a model of the extensive variability in genome dynamics among eukaryotes

Giardiasis from a One Health Perspective

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