RESUMO
El término CRISPR, por su acrónimo en inglés refiere a Clustered Regularly Interspaced Short Palindromic Repeats, es decir, repeticiones palindrómicas cortas, agrupadas y regularmente esparcidas, por sus características en el genoma, pertenece naturalmente al sistema de defensa de bacterias y arqueas. Este ha sido adaptado biotecnológicamente para la edición del ADN de células eucariotas, incluso de células humanas. El sistema CRISPR-Cas para editar genes consta, en forma generalizada, de dos componentes: una proteína nucleasa (Cas) y un ARN guía (sgRNA). La simplicidad del complejo lo hace una herramienta molecular reprogramable capaz de ser dirigida y de editar cualquier sitio en un genoma conocido. Su principal foco son las terapias para enfermedades hereditarias monogénicas y para el cáncer. Sin embargo, además de editor de genes, la tecnología CRISPR se utiliza para edición epigenética, regulación de la expresión génica y método de diagnóstico molecular. Este artículo tiene por objetivo presentar una revisión de las aplicaciones de la herramienta molecular CRISPR-Cas, particularmente en el campo biomédico, posibles tratamientos y diagnósticos, y los avances en investigación clínica, utilizando terapia génica con CRISPR/Cas más relevantes hasta la fecha. (AU)
CRISPR are Clustered Regularly Interspaced Short Palindromic Repeats, which naturally belong to the defense system of bacteria and archaea. It has been biotechnologically adapted for editing the DNA of eukaryotic cells, including human cells. The CRISPR-Cas system for editing genes generally consists of two components, a nuclease protein (Cas) and a guide RNA (sgRNA). The simplicity of the complex makes it a reprogrammable molecular tool capable of being targeted and editing any site in a known genome. Its main focus is therapies for monogenic inherited diseases and cancer. However, in addition to gene editor, CRISPR technology is used for epigenetic editing, regulation of gene expression, and molecular diagnostic methods. This article aims to present a review of the applications of the CRISPR-Cas molecular tool, particularly in the biomedical field, possible treatments and diagnoses, and the advances in clinical research, using the most relevant CRISPR-Cas gene therapy to date. (AU)
Assuntos
Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Sistemas CRISPR-Cas/genética , Biotecnologia , Terapia Genética/métodos , Expressão Gênica , Genoma Humano/genética , Regulação da Expressão Gênica , Epigenômica/tendências , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/uso terapêutico , Doenças Genéticas Inatas/terapia , Neoplasias/terapiaRESUMO
CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Farmacorresistência Bacteriana/genética , Endodesoxirribonucleases/genética , Edição de Genes/métodos , Vírus de RNA/genética , beta-Lactamases/farmacologia , DNA de Cadeia Simples/genética , Dengue/virologia , Vírus da Dengue/genética , Corantes Fluorescentes , Vírus Hantaan/genética , Humanos , Técnicas de Diagnóstico Molecular , Vírus de RNA/patogenicidade , RNA Viral/genética , Zika virus/genéticaRESUMO
Abstract: The growth factor receptor c-kit (CD117) is expressed in immature T-cells and in some advanced forms of mycosis fungoides. c-kit gene mutation results in unrestricted neoplastic proliferation. We aimed to detect by PCR the most frequent exon mutations in seventeen plaque-stage MF patients, in their perilesional skin and in healthy skin donors. We secondarily evaluated CD117 expression by immunohistochemistry in plaque-stage and tumor-stage MF. We detected no mutation in c-kit gene and low CD117 expression was confirmed on atypical cells in one patient. Complete c-kit exon and intron sequences should be assessed and more sensitive sequencing method could be also applied.
Assuntos
Humanos , Masculino , Feminino , Idoso , Éxons/genética , Micose Fungoide/genética , Proteínas Proto-Oncogênicas c-kit/genética , Mutação/genética , Imuno-Histoquímica , Estudos de Casos e Controles , Expressão Gênica , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
The growth factor receptor c-kit (CD117) is expressed in immature T-cells and in some advanced forms of mycosis fungoides. c-kit gene mutation results in unrestricted neoplastic proliferation. We aimed to detect by PCR the most frequent exon mutations in seventeen plaque-stage MF patients, in their perilesional skin and in healthy skin donors. We secondarily evaluated CD117 expression by immunohistochemistry in plaque-stage and tumor-stage MF. We detected no mutation in c-kit gene and low CD117 expression was confirmed on atypical cells in one patient. Complete c-kit exon and intron sequences should be assessed and more sensitive sequencing method could be also applied.
