Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis
ilustraciones, diagramas, tablas
- Autores:
-
Gutiérrez León, Jesús Esteban
- Tipo de recurso:
- Fecha de publicación:
- 2024
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/86807
- Palabra clave:
- 570 - Biología::572 - Bioquímica
570 - Biología::579 - Historia natural microorganismos, hongos, algas
VIROSIS
ESTREPTOCOCOS
REACCION EN CADENA DE LA POLIMERASA
Virus diseases
Streptococcus
Polimerase chain reaction
CRISPR-Cas9
Leishmania braziliensis
Anticuerpos policlonales IgY
T7 RNA Polimerasa
NMNAT
Estrés oxidativo
CRISPR-Cas9
Leishmania braziliensis
Polyclonal IgY antibodies
T7 RNA Polymerase
NMNAT
Oxidative stress
- Rights
- openAccess
- License
- Atribución-NoComercial 4.0 Internacional
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oai:repositorio.unal.edu.co:unal/86807 |
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UNACIONAL2 |
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Universidad Nacional de Colombia |
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|
| dc.title.spa.fl_str_mv |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| dc.title.translated.eng.fl_str_mv |
Development of molecular tools for the implementation of the CRISPR-Cas9 system in Leishmania braziliensis |
| title |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| spellingShingle |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis 570 - Biología::572 - Bioquímica 570 - Biología::579 - Historia natural microorganismos, hongos, algas VIROSIS ESTREPTOCOCOS REACCION EN CADENA DE LA POLIMERASA Virus diseases Streptococcus Polimerase chain reaction CRISPR-Cas9 Leishmania braziliensis Anticuerpos policlonales IgY T7 RNA Polimerasa NMNAT Estrés oxidativo CRISPR-Cas9 Leishmania braziliensis Polyclonal IgY antibodies T7 RNA Polymerase NMNAT Oxidative stress |
| title_short |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| title_full |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| title_fullStr |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| title_full_unstemmed |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| title_sort |
Desarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensis |
| dc.creator.fl_str_mv |
Gutiérrez León, Jesús Esteban |
| dc.contributor.advisor.spa.fl_str_mv |
Contreras Rodríguez, Luis Ernesto Téllez Meneses, Jair Alexander |
| dc.contributor.author.spa.fl_str_mv |
Gutiérrez León, Jesús Esteban |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::572 - Bioquímica 570 - Biología::579 - Historia natural microorganismos, hongos, algas |
| topic |
570 - Biología::572 - Bioquímica 570 - Biología::579 - Historia natural microorganismos, hongos, algas VIROSIS ESTREPTOCOCOS REACCION EN CADENA DE LA POLIMERASA Virus diseases Streptococcus Polimerase chain reaction CRISPR-Cas9 Leishmania braziliensis Anticuerpos policlonales IgY T7 RNA Polimerasa NMNAT Estrés oxidativo CRISPR-Cas9 Leishmania braziliensis Polyclonal IgY antibodies T7 RNA Polymerase NMNAT Oxidative stress |
| dc.subject.lemb.spa.fl_str_mv |
VIROSIS ESTREPTOCOCOS REACCION EN CADENA DE LA POLIMERASA |
| dc.subject.lemb.eng.fl_str_mv |
Virus diseases Streptococcus Polimerase chain reaction |
| dc.subject.proposal.spa.fl_str_mv |
CRISPR-Cas9 Leishmania braziliensis Anticuerpos policlonales IgY T7 RNA Polimerasa NMNAT Estrés oxidativo |
| dc.subject.proposal.eng.fl_str_mv |
CRISPR-Cas9 Leishmania braziliensis Polyclonal IgY antibodies T7 RNA Polymerase NMNAT Oxidative stress |
| description |
ilustraciones, diagramas, tablas |
| publishDate |
2024 |
| dc.date.accessioned.none.fl_str_mv |
2024-09-09T16:26:35Z |
| dc.date.available.none.fl_str_mv |
2024-09-09T16:26:35Z |
| dc.date.issued.none.fl_str_mv |
2024 |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Maestría |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/masterThesis |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/TM |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unal.edu.co/handle/unal/86807 |
| dc.identifier.instname.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.identifier.reponame.spa.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.unal.edu.co/ |
| url |
https://repositorio.unal.edu.co/handle/unal/86807 https://repositorio.unal.edu.co/ |
| identifier_str_mv |
Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
| dc.language.iso.spa.fl_str_mv |
spa |
| language |
spa |
| dc.relation.references.spa.fl_str_mv |
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Atribución-NoComercial 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Contreras Rodríguez, Luis Ernesto5d81511ea0a525c0165694621678d9c4Téllez Meneses, Jair Alexanderd50ed986507359558c5372c33a4d1190Gutiérrez León, Jesús Esteban264107d9eaaf3e9fe0e9e21cdd205f462024-09-09T16:26:35Z2024-09-09T16:26:35Z2024https://repositorio.unal.edu.co/handle/unal/86807Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, diagramas, tablasLas Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas (CRISPR) junto con sus proteínas asociadas (Cas), constituyen un sistema de defensa adaptativo procariota para contrarrestar infecciones virales. El sistema se ha aprovechado como una herramienta de edición génica programable, posibilitando diversos estudios y aplicaciones biotecnológicas, incluyendo la edición génica de organismos patogénicos como Leishmania y Trypanosoma. En Leishmania, el sistema CRISPR-Cas9 ha permitido caracterizar diversos genes de virulencia, así como la obtención de parásitos atenuados con potencial vacunal. Sin embargo, su implementación aún no ha sido reportada para este parásito en Colombia. El presente trabajó abordó el desarrollo de herramientas relacionadas con la implementación del sistema CRISPR-Cas9 en L. braziliensis, una de las especies circulantes en nuestro país. Específicamente, se emprendió la producción de anticuerpos policlonales aviares (IgY) anti-SpCas9, utilizando como antígeno la proteína Cas9 recombinante de Streptococcus pyogenes (SpCas9-6xHis). Estos anticuerpos resultaron ser sensibles, específicos y útiles para inmunodetectar la proteína SpCas9 en promastigotes de L. braziliensis transfectados con el plásmido pTB007 Viannia, el cual codifica para esta proteína. Adicionalmente, se abordó la expresión y purificación de la enzima T7 RNA Polimerasa (6xHis-T7RNAP) desde Escherichia coli, resultando útil para sintetizar ARN guías (sgRNA) y preparar complejos ribonucleoproteicos (SpCas9-sgRNA) funcionales, capaces de efectuar cortes de ADN in vitro. Por último, se implementó el sistema CRISPR-Cas9 para realizar edición génica sobre la nicotinamida mononucleótido adenililtransferasa de L. braziliensis (LbNMNAT), que participa en la síntesis del NAD, insertando la secuencia CfPGKB5’-mCherry en el extremo 5’ del gen de interés en parásitos que expresan SpCas9 y T7RNAP. Los análisis funcionales del gen revelaron un aumento de sus niveles de expresión, mientras que los ensayos de susceptibilidad ante estrés oxidativo mostraron mayores valores IC50 en los parásitos editados en comparación con muestras control, lo que sugiere que la síntesis del NAD puede estar relacionada con fenotipos fármaco-resistentes. De esta manera, se obtuvieron herramientas que facilitan la implementación del sistema CRISPR-Cas9 en L. braziliensis, un patógeno de interés para la salud pública del país. Así mismo, se demostró la posibilidad de aprovechar sistemas avanzados de biología molecular para estudiar genes relacionados con la síntesis del NAD en el contexto de enfermedades tropicales desatendidas como la Leishmaniasis (Texto tomado de la fuente).Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas), constitute a prokaryotic adaptive defense system to counter viral infections. The system has been harnessed as a programmable gene-editing tool, enabling diverse biotechnological studies and applications, including gene editing of pathogenic organisms such as Leishmania and Trypanosoma. In Leishmania, the CRISPR-Cas9 system has made it possible to characterize several virulence genes and obtain attenuated parasites with vaccine potential. However, its implementation has not yet been reported for this parasite in Colombia. In this sense, the present work addressed the development of tools related to implementing the CRISPR-Cas9 system in L. braziliensis, one of the species circulating in our country. Specifically, the production of anti-SpCas9 avian polyclonal antibodies (IgY) was undertaken, using the recombinant Cas9 protein from Streptococcus pyogenes (SpCas9-6xHis) as antigen. The antibodies were sensitive, specific, and useful to immunodetect SpCas9 protein in L. braziliensis promastigotes transfected with plasmid pTB007 Viannia, which encodes for this protein. Additionally, the expression and purification of the enzyme T7 RNA Polymerase (6xHis-T7RNAP) from Escherichia coli were addressed, proving useful for synthesizing guide RNA (sgRNA) and preparing functional ribonucleoprotein complexes (SpCas9-sgRNA), capable of performing in vitro DNA cleavage. Finally, the gene coding for the L. braziliensis nicotinamide mononucleotide adenylyltransferase (LbNMNAT), which is involved in NAD synthesis, was targeted for editing. Using parasites expressing SpCas9 and T7RNAP proteins, the sequence CfPGKB5'-mCherry was inserted at the 5' end of the gene of interest, favoring the increase of LbNMNAT expression levels. Phenotypic assays of susceptibility to oxidative stress revealed higher IC50 values in the edited parasites compared to control samples, suggesting that NAD synthesis is related to drug tolerance. In this way, several tools were obtained that facilitate the implementation of the CRISPR-Cas9 system in L. braziliensis, a pathogen of public health interest in Colombia, demonstrating the possibility of taking advantage of advanced molecular biology systems to study genes related to NAD synthesis in the context of neglected tropical diseases such as Leishmaniasis.MaestríaMagíster en Ciencias - BioquímicaLa Figura 5.1 presenta la metodología general abordada en este trabajo. Inicialmente, se realizó la producción de anticuerpos policlonales tipo IgY dirigidos contra SpCas9 en modelos aviares, los cuales fueron caracterizados mediante ensayos de Western blot y ELISA, así como su uso sobre muestras biológicas de L. braziliensis (cepa de referencia MHOM/BR/75/M2903, denominada LbWT, y cepa que expresa de manera constitutiva SpCas9 y T7RNAP, denominada LbCas9T7). Seguidamente, se evaluó el ensamblaje de complejos ribonucleoproteicos, utilizando la proteína SpCas9-6xHis y el sgRNA sintetizado in vitro con la enzima 6xHis-T7RNAP. Luego, se ensayó la actividad nucleasa de los complejos ensamblados sobre ADN plasmídico y un producto de PCR, contra el gen Lbnmnat. Finalmente, se implementó el sistema CRISPR-Cas9 para editar mediante etiquetado (o tagging) el gen Lbnmnat en promastigotes de la cepa LbCas9T7, lo que permitió obtener una cepa editada, denominada LbNTag. Se estandarizaron los protocolos de transfección mediante electroporación para una eficiente entrega de casetes de reparación y plantillas de sgRNA. Mediante ensayos de PCR diagnósticos, se evaluó el éxito de la edición génica en los sitios genómicos de interés. Mediante ensayo de secuenciamiento Sanger, se comprobó la inserción de la plantilla de reparación en el sitio de interés. Finalmente, se caracterizó el efecto a nivel fenotípico de la edición realizada sobre la cepa editada, evaluando su respuesta a agentes causantes de estrés oxidativo como H2O2 y Sb3+.Bioquímica y Biología Molecular de Parásitosxix, 133 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - BioquímicaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::572 - Bioquímica570 - Biología::579 - Historia natural microorganismos, hongos, algasVIROSISESTREPTOCOCOSREACCION EN CADENA DE LA POLIMERASAVirus diseasesStreptococcusPolimerase chain reactionCRISPR-Cas9Leishmania braziliensisAnticuerpos policlonales IgYT7 RNA PolimerasaNMNATEstrés oxidativoCRISPR-Cas9Leishmania braziliensisPolyclonal IgY antibodiesT7 RNA PolymeraseNMNATOxidative stressDesarrollo de herramientas moleculares para la implementación del sistema CRISPR-Cas9 en Leishmania braziliensisDevelopment of molecular tools for the implementation of the CRISPR-Cas9 system in Leishmania braziliensisTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMAdaui, V., Kröber-Boncardo, C., Brinker, C., Zirpel, H., Sellau, J., Arévalo, J., Dujardin, J. 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Analytical Biochemistry, 236(2), 302–308. https://doi.org/10.1006/ABIO.1996.0171EstudiantesInvestigadoresLICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/86807/3/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD53ORIGINAL1024588797.2024.pdf1024588797.2024.pdfTesis de Maestría en Ciencias Bioquímicaapplication/pdf9736754https://repositorio.unal.edu.co/bitstream/unal/86807/4/1024588797.2024.pdffb1ff686b186c678897c0dbbe86bd7a9MD54THUMBNAIL1024588797.2024.pdf.jpg1024588797.2024.pdf.jpgGenerated Thumbnailimage/jpeg5226https://repositorio.unal.edu.co/bitstream/unal/86807/5/1024588797.2024.pdf.jpg60b17eb6ad3a76cfe9e342b0f1373975MD55unal/86807oai:repositorio.unal.edu.co:unal/868072024-09-09 23:05:08.463Repositorio Institucional Universidad Nacional de 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