Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: Proof of concept

Kangussu-Marcolino, Mônica Mendes; Cunha, Ana Paula; Ávila, Andréa Rodrigues; Herman, J.P.; DaRocha, Wanderson D.

doi:10.1016/j.molbiopara.2015.01.001

Cited by 12 publications

(8 citation statements)

References 16 publications

(29 reference statements)

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“…Additionally, new technologies are now available to facilitate genome editing in T. cruzi , such as the Cre-recombinases and the CRISPR-Cas9 system. These genetic manipulation strategies have highly effective efficiency in different organisms and now were successfully adapted to disrupt genes from T. cruzi [145–147]. Furthermore the CRISPR-Cas9 system was recently used for endogenous tagging of proteins in T. cruzi which proved that this system is not limited to loss-of-function and made the localization/visualization of proteins from inside the parasite possible [148].…”

Section: Discussionmentioning

confidence: 99%

A Brief View of the Surface Membrane Proteins fromTrypanosoma cruzi

Pech-Canul

Monteón

Solís-Oviedo

2017

Journal of Parasitology Research

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Trypanosoma cruzi is the causal agent of Chagas' disease which affects millions of people around the world mostly in Central and South America. T. cruzi expresses a wide variety of proteins on its surface membrane which has an important role in the biology of these parasites. Surface molecules of the parasites are the result of the environment to which the parasites are exposed during their life cycle. Hence, T. cruzi displays several modifications when they move from one host to another. Due to the complexity of this parasite's cell surface, this review presents some membrane proteins organized as large families, as they are the most abundant and/or relevant throughout the T. cruzi membrane.

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Section: Discussionmentioning

confidence: 99%

A Brief View of the Surface Membrane Proteins fromTrypanosoma cruzi

Pech-Canul

Monteón

Solís-Oviedo

2017

Journal of Parasitology Research

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“…Inducible systems using destabilization domains of dihydrofolate reductase (DDD), or the rapamycin binding protein (ddFKBP), were only used to either create suicidal T. cruzi strains (Ma et al, 2015), or did not mediate the efficient knockdown of the genes (Burle-Caldas Gde et al, 2015). Inducible expression of dimerizable CRE recombinase (DiCRE system) was also tried in T. cruzi but has been only used for removal of exogenous selectable markers from the parasite's genome with limited success (Kangussu-Marcolino et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi

Lander

Cruz‐Bustos

Docampo

2020

Front. Cell. Infect. Microbiol.

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Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.

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“…An alternative to the mentioned traditional methods to evaluate essentially is the use of inducible approaches such as Di-Cre or the DHFR degradation domain, or genome edition tools such as CRISPR-Cas9 or Zinc-finger endonucleases. These systems have been adapted to T. cruzi in the last 5 years (Kangussu-Marcolino et al, 2014; Peng et al, 2014; Lander et al, 2015; Ma et al, 2015; Burle-Caldas et al, 2017), although recent work has focused on the optimization of different CRISPR-Cas9 configurations (e.g., Beneke et al, 2017; Lander et al, 2017; Soares Medeiros et al, 2017; Burle-Caldas et al, 2018; Costa et al, 2018). The future widespread use of CRISPR-Cas9 will help overcome the current technical limitations for the genetic manipulation of T. cruzi .…”

Section: Identification Of New Treatments For Chagas' Diseasementioning

confidence: 99%

Discovery and Genetic Validation of Chemotherapeutic Targets for Chagas' Disease

Osorio-Méndez

Cevallos

2019

Front. Cell. Infect. Microbiol.

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There is an urgent need to develop new treatments for Chagas' disease. To identify drug targets, it is important to understand the basic biology of Trypanosoma cruzi, in particular with respect to the biological pathways or proteins that are essential for its survival within the host. This review provides a streamlined approach for identifying drug targets using freely available chemogenetic databases and outlines the relevant characteristics of an ideal chemotherapeutic target. Among those are their essentiality, druggability, availability of structural information, and selectivity. At the moment only 16 genes have been found as essential by gene disruption in T. cruzi. At the TDR Targets database, a chemogenomics resource for neglected diseases, information about published structures for these genes was only found for three of these genes, and annotation of validated inhibitors was found in two. These inhibitors have activity against the parasitic stages present in the host. We then analyzed three of the pathways that are considered promising in the search for new targets: (1) Ergosterol biosynthesis, (2) Resistance to oxidative stress, (3) Synthesis of surface glycoconjugates. We have annotated all the genes that participate in them, identified those that are considered as druggable, and incorporated evidence from either Trypanosoma brucei, and Leishmania spp. that supports the hypothesis that these pathways are essential for T. cruzi survival.

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Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: Proof of concept

Cited by 12 publications

References 16 publications

A Brief View of the Surface Membrane Proteins fromTrypanosoma cruzi

A Brief View of the Surface Membrane Proteins fromTrypanosoma cruzi

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi

Discovery and Genetic Validation of Chemotherapeutic Targets for Chagas' Disease

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