CREditing: a tool for gene tuning in Trypanosoma cruzi

The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insert...

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Autores:: Pacheco-Lugo, Lisandro A.
Sáenz-García, José L.
Díaz-Olmos, Yirys
Netto-Costa, Rodrigo
Brant, Rodrigo S. C.
DaRocha, Wanderson D.

Tipo de recurso:

Fecha de publicación:: 2020

Institución:: Universidad Simón Bolívar

Repositorio:: Repositorio Digital USB

Idioma:: eng

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dc.title.eng.fl_str_mv	CREditing: a tool for gene tuning in Trypanosoma cruzi
title	CREditing: a tool for gene tuning in Trypanosoma cruzi
spellingShingle	CREditing: a tool for gene tuning in Trypanosoma cruzi Trypanosoma cruzi CREditing CRE recombinase CRE-lox Conditional gene deletion Gene activation
title_short	CREditing: a tool for gene tuning in Trypanosoma cruzi
title_full	CREditing: a tool for gene tuning in Trypanosoma cruzi
title_fullStr	CREditing: a tool for gene tuning in Trypanosoma cruzi
title_full_unstemmed	CREditing: a tool for gene tuning in Trypanosoma cruzi
title_sort	CREditing: a tool for gene tuning in Trypanosoma cruzi
dc.creator.fl_str_mv	Pacheco-Lugo, Lisandro A. Sáenz-García, José L. Díaz-Olmos, Yirys Netto-Costa, Rodrigo Brant, Rodrigo S. C. DaRocha, Wanderson D.
dc.contributor.author.none.fl_str_mv	Pacheco-Lugo, Lisandro A. Sáenz-García, José L. Díaz-Olmos, Yirys Netto-Costa, Rodrigo Brant, Rodrigo S. C. DaRocha, Wanderson D.
dc.subject.eng.fl_str_mv	Trypanosoma cruzi CREditing CRE recombinase CRE-lox Conditional gene deletion Gene activation
topic	Trypanosoma cruzi CREditing CRE recombinase CRE-lox Conditional gene deletion Gene activation
description	The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tdTomato (tandem dimer fluorescent protein) reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of function approaches.
publishDate	2020
dc.date.accessioned.none.fl_str_mv	2020-08-25T22:11:02Z
dc.date.available.none.fl_str_mv	2020-08-25T22:11:02Z
dc.date.issued.none.fl_str_mv	2020
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dc.type.spa.spa.fl_str_mv	Artículo científico
dc.identifier.issn.none.fl_str_mv	00207519
dc.identifier.uri.none.fl_str_mv	https://hdl.handle.net/20.500.12442/6343
dc.identifier.doi.none.fl_str_mv	https://doi.org/10.1016/j.ijpara.2020.06.010
identifier_str_mv	00207519
url	https://hdl.handle.net/20.500.12442/6343 https://doi.org/10.1016/j.ijpara.2020.06.010
dc.language.iso.eng.fl_str_mv	eng
language	eng
dc.rights.none.fl_str_mv	Attribution-NonCommercial-NoDerivatives 4.0 Internacional
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eu_rights_str_mv	openAccess
dc.format.mimetype.spa.fl_str_mv	pdf
dc.publisher.eng.fl_str_mv	Elsevier Australian Society for Parasitology
dc.source.eng.fl_str_mv	International Journal for Parasitology
institution	Universidad Simón Bolívar
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spelling	Pacheco-Lugo, Lisandro A.3d97b683-f7ae-40dd-86f5-c5ffbd48e0c3Sáenz-García, José L.193e1f04-03ee-4684-8c38-ca9b96297b52Díaz-Olmos, Yirysdf23bcb1-18f3-44a7-ae31-f374fb4947d6Netto-Costa, Rodrigo5477482d-db5c-46aa-a01a-31b4aa7abf5eBrant, Rodrigo S. C.602ebc0c-8323-43f8-9f89-1edcf3099e4dDaRocha, Wanderson D.a9ad1c3a-be1c-40d9-b611-9474f6cb54b42020-08-25T22:11:02Z2020-08-25T22:11:02Z202000207519https://hdl.handle.net/20.500.12442/6343https://doi.org/10.1016/j.ijpara.2020.06.010The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tdTomato (tandem dimer fluorescent protein) reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of function approaches.pdfengElsevierAustralian Society for ParasitologyAttribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2International Journal for ParasitologyTrypanosoma cruziCREditingCRE recombinaseCRE-loxConditional gene deletionGene activationCREditing: a tool for gene tuning in Trypanosoma cruziinfo:eu-repo/semantics/articleArtículo científicohttp://purl.org/coar/version/c_71e4c1898caa6e32http://purl.org/coar/resource_type/c_2df8fbb1Albert, H., Dale, E.C., Lee, E., Ow, D.W., 1995. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. Cell Mol. Biol. 7, 649–659.Barrett, B., LaCount, D.J., Donelson, J.E., 2004. Trypanosoma brucei: a first-generation CRE-loxP site-specific recombination system. Exp. Parasitol. 106, 37–44. https://doi.org/10.1016/j.exppara.2004.01.004Bergemann, J., Kühlcke, K., Fehse, B., Ratz, I., Ostertag, W., Lother, H., 1995. Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination. Nucleic Acids Res. 23, 4451–4456.Burle-Caldas, G.A., Soares-Simões, M., Lemos-Pechnicki, L., DaRocha, W.D., Teixeira, S.M.R., 2018. Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants. Int. J. Parasitol. 48, 591–596. https://doi.org/10.1016/j.ijpara.2018.02.002Burle-Caldas Gde A, Grazielle-Silva V, Laibida LA, DaRocha WD, Teixeira SM, 2015. Expanding the tool box for genetic manipulation of Trypanosoma cruzi. Mol Biochem Parasitol. 203, 25-33. https://doi.org/10.1016/j.molbiopara.2015.10.004.Chen, C.-M., Behringer, R.R., 2001. CREating breakthroughs. Nat. Biotechnol. 19, 921–922. https://doi.org/10.1038/nbt1001-921Chiurillo, M.A., Lander, N., Bertolini, M.S., Storey, M., Vercesi, A.E., Docampo, R., 2017. Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of Trypanosoma cruzi. mBio 8, e00574-17. https://doi.org/10.1128/mBio.00574-17Contreras, V.T., Salles, J.M., Thomas, N., Morel, C.M., Goldenberg, S., 1985. In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol. Biochem. Parasitol. 16, 315–327.Cruz-Bustos, T., Potapenko, E., Storey, M., Docampo, R., 2018. An Intracellular Ammonium Transporter Is Necessary for Replication, Differentiation, and Resistance to Starvation and Osmotic Stress in Trypanosoma cruzi. mSphere 3, e00377-17. https://doi.org/10.1128/mSphere.00377-17Damasceno, J.D., Reis-Cunha, J., Crouch, K., Lapsley, C., Tosi, L.R.O. Bartholomeu, D., McCulloch, R., 2020. Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication. PLoS Genet. 16(7): e1008828. http://doi: 10.1371/journal.pgen.1008828.DaRocha, W.D., Otsu, K., Teixeira, S.M.R., Donelson, J.E., 2004a. Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi. Mol. Biochem. Parasitol. 133, 175–186.DaRocha, W.D., Silva, R.A., Bartholomeu, D.C., Pires, S.F., Freitas, J.M., Macedo, A.M., Vazquez, M.P., Levin, M.J., Teixeira, S.M.R., 2004b. Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols. Parasitol. Res. 92, 113–120. https://doi.org/10.1007/s00436-003-1004-5Deora, A.A., Diaz, F., Schreiner, R., Rodriguez-Boulan, E., 2007. Efficient Electroporation of DNA and Protein into Confluent and Differentiated Epithelial Cells in Culture. Traffic 8, 1304–1312. https://doi.org/10.1111/j.1600-0854.2007.00617.xFuruhata, Y., Sakai, A., Murakami, T., Morikawa, M., Nakamura, C., Yoshizumi, T., Fujikura, U., Nishida, K., Kato, Y., 2019. A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall. Sci. Rep. 9. https://doi.org/10.1038/s41598-018-38119-9González, L., García-Huertas, P., Triana-Chávez, O., García, G.A., Murta, S.M.F., Mejía-Jaramillo, A.M., 2017. Aldo-keto reductase and alcohol dehydrogenase contribute to benznidazole natural resistance in Trypanosoma cruzi: Benznidazole natural resistance in Trypanosoma cruzi. Mol. Microbiol. 106, 704–718. https://doi.org/10.1111/mmi.13830Hall, B.S., Bot, C., Wilkinson, S.R., 2011. Nifurtimox Activation by Trypanosomal Type I Nitroreductases Generates Cytotoxic Nitrile Metabolites. J. Biol. Chem. 286, 13088–13095. https://doi.org/10.1074/jbc.M111.230847Jullien, N., Sampieri, F., Enjalbert, A., Herman, J., 2003. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, 131e–1131. https://doi.org/10.1093/nar/gng131Jullien, N., Goddard, I., Selmi-Ruby, S., Fina, J.-L., Cremer, H., Herman, J.-P., 2007. Conditional Transgenesis Using Dimerizable Cre (DiCre). PLoS ONE 2, e1355. https://doi.org/10.1371/journal.pone.0001355Kangussu-Marcolino, M.M., Cunha, A.P., Avila, A.R., Herman, J.-P., DaRocha, W.D., 2014. Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: Proof of concept. Mol. Biochem. Parasitol. 198, 71–74. https://doi.org/10.1016/j.molbiopara.2015.01.001Lander, N., Chiurillo, M., Vercesi, A., Docampo, R., 2017. Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi. BIO-Protoc. 7. https://doi.org/10.21769/BioProtoc.2299Lander, N., Chiurillo, M.A., Storey, M., Vercesi, A.E., Docampo, R., 2016. CRISPR/Cas9- mediated endogenous C-terminal tagging of Trypanosoma cruzi genes reveals the acidocalcisome localization of the inositol 1,4,5-trisphosphate receptor. J. Biol. Chem. 291, 25505–25515. https://doi.org/10.1074/jbc.M116.749655Lander, N., Li, Z.-H., Niyogi, S., Docampo, R., 2015. CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment. mBio 6, e01012-15. https://doi.org/10.1128/mBio.01012-15Laverrière, M., Cazzulo, J.J., Alvarez, V.E., 2012. Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation. Cell Death Differ. 19, 1358–1369. https://doi.org/10.1038/cdd.2012.12Lee, G., Saito, I., 1998. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216, 55–65. https://doi.org/10.1016/S0378-1119(98)00325-4Lewandoski, M., 2001. Mouse genomic technologies: conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743–755. https://doi.org/10.1038/35093537 Liu, J., Willet, S.G., Bankaitis, E.D., Xu, Y., Wright, C.V.E., Gu, G., 2013. Non-parallel recombination limits cre-loxP-based reporters as precise indicators of conditional genetic manipulation: Cre-Recombinations are Non-Parallel Events. genesis 51, 436–442. https://doi.org/10.1002/dvg.22384Loonstra, A., Vooijs, M., Beverloo, H.B., Allak, B.A., van Drunen, E., Kanaar, R., Berns, A., Jonkers, J., 2001. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 98, 9209–9214. https://doi.org/10.1073/pnas.161269798Marchetti, M.A., Tschudi, C., Kwon, H., Wolin, S.L., Ullu, E., 2000. Import of proteins into the trypanosome nucleus and their distribution at karyokinesis. J. Cell Sci. 113 ( Pt 5), 899–906.Olmo, F., Costa, F.C., Mann, G.S., Taylor, M.C., Kelly, J.M., 2018. Optimising genetic transformation of Trypanosoma cruzi using hydroxyurea-induced cell-cycle synchronisation. Mol. Biochem. Parasitol. 226, 34–36. https://doi.org/10.1016/j.molbiopara.2018.07.002Pacheco-Lugo, L., Díaz-Olmos, Y., Sáenz-García, J., Probst, C.M., DaRocha, W.D., 2017. Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection. Parasitol. Int. 66, 236–239. https://doi.org/10.1016/j.parint.2017.01.019Padmanabhan, P.K., Polidoro, R.B., Barteneva, N.S., Gazzinelli, R.T., Burleigh, B.A., 2014. Transient transfection and expression of foreign and endogenous genes in the intracelular stages of Trypanosoma cruzi. Mol. Biochem. Parasitol. 198, 100–103. https://doi.org/10.1016/j.molbiopara.2015.02.001Peng, D., Kurup, S.P., Yao, P.Y., Minning, T.A., Tarleton, R.L., 2015. CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. mBio 6, e02097-02014. https://doi.org/10.1128/mBio.02097-14Piacenza, L., Irigoín, F., Alvarez, M.N., Peluffo, G., Taylor, M.C., Kelly, J.M., Wilkinson, S.R., Radi, R., 2007. Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi : cytoprotective action of mitochondrial iron superoxide dismutase overexpression. Biochem. J. 403, 323–334. https://doi.org/10.1042/BJ20061281Romagnoli, B.A.A., Picchi, G.F.A., Hiraiwa, P.M., Borges, B.S., Alves, L.R., Goldenberg, S., 2018. Improvements in the CRISPR/Cas9 system for high efficiency gene disruption in Trypanosoma cruzi. Acta Trop. 178, 190–195. https://doi.org/10.1016/j.actatropica.2017.11.013Santos, R.E.R.S., Silva, G.L.A., Santos, E.V., Duncan, S.M., Mottram, J.C., Damasceno, J.D., Tosi,L.R.O., 2017. A DiCre recombinase-based system for inducible expression in Leishmania major. Mol. Biochem. Parasitol. 216, 45–48. https://doi.org/10.1016/j.molbiopara.2017.06.006Sauer, B., 1998. Inducible gene targeting in mice using the Cre/lox system. Methods San Diego Calif 14, 381–392. https://doi.org/10.1006/meth.1998.0593Scahill, M.D., Pastar, I., Cross, G.A.M., 2008. CRE recombinase-based positive-negative selection systems for genetic manipulation in Trypanosoma brucei. Mol. Biochem. Parasitol. 157:73-82. https://doi.org/10.1016/j.molbiopara.2007.10.003.Schmidt, E.E., Taylor, D.S., Prigge, J.R., Barnett, S., Capecchi, M.R., 2000. Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc. Natl. Acad. Sci. 97, 13702–13707. https://doi.org/10.1073/pnas.240471297Schumann Burkard, G., Jutzi, P., Roditi, I., 2011. Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Mol. Biochem. Parasitol. 175, 91–94. https://doi.org/10.1016/j.molbiopara.2010.09.002Silver, D.P., Livingston, D.M., 2001. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol. Cell 8, 233–243.Soares Medeiros, L.C., South, L., Peng, D., Bustamante, J.M., Wang, W., Bunkofske, M., Perumal, N., Sanchez-Valdez, F., Tarleton, R.L., 2017. Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins. mBio 8, e01788-17. https://doi.org/10.1128/mBio.01788-17Song, Y., Yuan, L., Wang, Y., Chen, M., Deng, J., Lv, Q., Sui, T., Li, Z., Lai, L., 2016. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system. Cell. Mol. Life Sci. 73, 2959–2968. https://doi.org/10.1007/s00018-016-2143-zTaylor, M.C., Kelly, J.M., 2006. pTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi. BMC Biotechnol. 6, 32. https://doi.org/10.1186/1472-6750-6-32Testa, G., Stewart, A.F., 2000. Cre ating a trans lox ation: Engineering interchromosomal translocations in the mouse. EMBO Rep. 1, 120–121. https://doi.org/10.1093/embo-reports/kvd035Tronche, F., Casanova, E., Turiault, M., Sahly, I., Kellendonk, C., 2002. When reverse genetics meets physiology: the use of site-specific recombinases in mice. FEBS Lett. 529, 116–121.Wyllie, S., Patterson, S., Fairlamb, A.H., 2013. Assessing the Essentiality of Leishmania donovani Nitroreductase and Its Role in Nitro Drug Activation. Antimicrob. 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CREditing: a tool for gene tuning in Trypanosoma cruzi

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