Knockout del gen BCY84_00258 en Epimastigotes de Trypanosoma Cruzi DM28C empleando la técnica de edición génica CRISPR-CAS9
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2024
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
En el marco de la problemática de la enfermedad de Chagas, causada por el
parásito Trypanosoma cruzi y el incremento en la incidencia de casos en América
Latina en conjunto con el hecho de que únicamente existen dos tratamientos para
el control de la enfermedad (Nufurtimox y Benznidazol), motivó la realización de un
estudio de edición genética en genes constitutivos de Trypanosoma cruzi que al
evaluarse su función y las consecuencias de su silenciamiento o knockout como
forma de sentar las bases para estudios posteriores en los que se investiguen
formas de inhibir el metabolismo parasitario y por consiguiente, la muerte del agente
etiológico de la enfermedad de Chagas.
Es por esto por lo que como un primer acercamiento a determinar el efecto del
knockout del gen BCY84_00258 en el parasito, se planteó como objetivo principal
de este proyecto llevar a cabo el cleavage in vitro del gen BCY84_00258 en
epimastigotes de Trypanosoma cruzi utilizando la técnica de edición génica
CRISPR-Cas9.
Esta investigación se enfocó en comprender el papel de este gen en la biología del
parásito y evaluar su potencial como blanco para el desarrollo de nuevas estrategias
terapéuticas contra la enfermedad de Chagas. La metodología empleada consistió
en generación de un vector plasmídico el cual contuviese la información genética
necesaria para la síntesis de las moléculas involucradas en el sistema CRISPRCas, para esto se alinearon oligonucleotidos correspondientes a secuencias de
DNA de aproximadamente 20pb las cuales hacen parte de la secuencia del gen de
interés de forma que al ser transcritos permitan la formación de crRNA, a su vez,
aguas abajo de este se introduce una secuencia que codifique para el tracrRNA,
estos dos últimos forman el sgRNA necesario para dirigir la Cas9 al sitio especifico
de corte. La extracción de ADN genómico de T. cruzi cepa DM28C para la
amplificación e identificación del gen BCY84_00258, el cual funcionó como el DNA
template para la reacción de cleavage in vitro del gen en cuestión, seguido del
diseño y la producción de Cas9 de Staphylococcus aureus (SaCas9) recombinante
a partir de la clonación de bacterias E. coli Rosetta de un plásmido que contenga la
secuencia de dicha endonucleasa, su sobreexpresión, extracción y purificación,
garantizando la actividad de la enzima posterior al proceso.
Se logró generar un sgRNA para el gen BYC84_00258. Se amplificó un fragmento
de 292pb del gen BCY84_00258 con primers específicos para generar una
secuencia complementaria al sgRNA generado permitiendo el corte de dsDNA por
parte de la Cas9. La extracción y purificación de la Cas9 se realizó mediante FPLC,
seleccionando fracciones con un valor pico >50mAU como criterio de calidad. Se
llevó a cabo el cleavaje in vitro obteniéndose un corte parcial del DNA. Estos datos
resaltan el potencial del gen BCY_84_00258 como gen de interés biotecnológico
prometedor para el desarrollo de nuevas estrategias para la enfermedad de Chagas
como la búsqueda y evaluación de moléculas que inhiban la actividad del producto
proteico del gen que permitan su uso como terapia contra la enfermedad, abriendo
nuevas posibilidades en la lucha contra esta enfermedad endémica de la zona
tropical del continente americano.
In the context of the problem of Chagas disease, caused by the parasite Trypanosoma cruzi parasite and the increase in the incidence of cases in Latin America in conjunction with the fact that there are only two treatments to control the disease (Nufurtimox and Benznidazole ), motivated the carrying out of a genetic editing study on constituent genes of Trypanosoma cruzi that evaluated its function and the consequences of its silencing or knockout as a way of laying the foundations for subsequent studies in which ways to inhibit parasitic metabolism are investigated. and consequently, the death of the etiological agent of Chagas disease. This is why, as a first approach to determining the effect of the knockout of the BCY84_00258 gene in the parasite, the main objective of this project was to carry out the in vitro cleavage of the BCY84_00258 gene in epimastigotes of Trypanosoma cruzi using the CRISPR-Cas9 technique of gene editing. This research focused on understanding the role of this gene in the biology of the parasite and evaluating its potential as a target for the development of new therapeutic strategies against Chagas disease. The methodology used consisted of generating a plasmid vector which contained the genetic information necessary for the synthesis of the molecules involved in the CRISPR-Cas system. For this, oligonucleotides corresponding to DNA sequences of approximately 20bp were aligned using the ramp alignment technique, which are part of the sequence of the gene of interest so that when transcribed they allow the formation of crRNA, in turn, downstream of this a sequence is introduced that codes for the tracrRNA, the latter two form the sgRNA necessary to direct Cas9 to the specific site of court. The extraction of genomic DNA from T. cruzi strain DM28C for the amplification and identification of the gene BCY84_00258, which functioned as the DNA template for the in vitro cleavage reaction of the gene in question, followed by the design and production of Cas9 from Staphylococcus aureus (SaCas9) recombinant from the cloning of E. coli Rosetta bacteria of a plasmid that contains the sequence of said endonuclease, its overexpression, extraction and purification, guaranteeing the activity of the enzyme after the process. It was possible to generate an sgRNA for the BYC84_00258 gene. A 292bp fragment of the BCY84_00258 gene was amplified with specific primers to generate a sequence complementary to the generated sgRNA allowing the cutting of dsDNA by Cas9. The extraction and purification of Cas9 was carried out by FPLC, selecting fractions with a peak value >50mAU as a quality criterion. Cleavage was carried out in vitro, obtaining a partial cut of the DNA. These data highlight the potential of the BCY_84_00258 gene as a gene of promising biotechnological interest for the development of new strategies for Chagas disease, such as the search and evaluation of molecules that inhibit the activity of the gene's protein product, allowing its use as therapy against the disease, opening new possibilities in the fight against this disease endemic to the tropical zone of the American continent.
In the context of the problem of Chagas disease, caused by the parasite Trypanosoma cruzi parasite and the increase in the incidence of cases in Latin America in conjunction with the fact that there are only two treatments to control the disease (Nufurtimox and Benznidazole ), motivated the carrying out of a genetic editing study on constituent genes of Trypanosoma cruzi that evaluated its function and the consequences of its silencing or knockout as a way of laying the foundations for subsequent studies in which ways to inhibit parasitic metabolism are investigated. and consequently, the death of the etiological agent of Chagas disease. This is why, as a first approach to determining the effect of the knockout of the BCY84_00258 gene in the parasite, the main objective of this project was to carry out the in vitro cleavage of the BCY84_00258 gene in epimastigotes of Trypanosoma cruzi using the CRISPR-Cas9 technique of gene editing. This research focused on understanding the role of this gene in the biology of the parasite and evaluating its potential as a target for the development of new therapeutic strategies against Chagas disease. The methodology used consisted of generating a plasmid vector which contained the genetic information necessary for the synthesis of the molecules involved in the CRISPR-Cas system. For this, oligonucleotides corresponding to DNA sequences of approximately 20bp were aligned using the ramp alignment technique, which are part of the sequence of the gene of interest so that when transcribed they allow the formation of crRNA, in turn, downstream of this a sequence is introduced that codes for the tracrRNA, the latter two form the sgRNA necessary to direct Cas9 to the specific site of court. The extraction of genomic DNA from T. cruzi strain DM28C for the amplification and identification of the gene BCY84_00258, which functioned as the DNA template for the in vitro cleavage reaction of the gene in question, followed by the design and production of Cas9 from Staphylococcus aureus (SaCas9) recombinant from the cloning of E. coli Rosetta bacteria of a plasmid that contains the sequence of said endonuclease, its overexpression, extraction and purification, guaranteeing the activity of the enzyme after the process. It was possible to generate an sgRNA for the BYC84_00258 gene. A 292bp fragment of the BCY84_00258 gene was amplified with specific primers to generate a sequence complementary to the generated sgRNA allowing the cutting of dsDNA by Cas9. The extraction and purification of Cas9 was carried out by FPLC, selecting fractions with a peak value >50mAU as a quality criterion. Cleavage was carried out in vitro, obtaining a partial cut of the DNA. These data highlight the potential of the BCY_84_00258 gene as a gene of promising biotechnological interest for the development of new strategies for Chagas disease, such as the search and evaluation of molecules that inhibit the activity of the gene's protein product, allowing its use as therapy against the disease, opening new possibilities in the fight against this disease endemic to the tropical zone of the American continent.
Descripción
Palabras clave
Trypanosoma cruzi, CRISPR-Cas9, Cleavage, BCY84_00258, Edición génica
