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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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El objetivo del ARN con CRISPR-Cas13

Omar O Abudayyeh1,2,3,4,5, Jonathan S Gootenberg1,2,3,4,6, Patrick Essletzbichler1,2,3,4

  • 1Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

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|October 5, 2017
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Los científicos diseñaron CRISPR-Cas13a para una manipulación precisa del ARN en las células de los mamíferos. Esta nueva herramienta ofrece un knockdown de ARN dirigido y un seguimiento de transcripción de células vivas con una especificidad mejorada en comparación con los métodos existentes.

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Área de la Ciencia:

  • Biología molecular
  • La genética
  • Biotecnología

Sus antecedentes:

  • Las herramientas moleculares para la manipulación y medición del ARN son limitadas.
  • Los métodos actuales como la interferencia de ARN tienen efectos fuera del objetivo.
  • La visualización del ARN a menudo requiere etiquetas exógenas.

Objetivo del estudio:

  • Diseñar el sistema CRISPR-Cas13a para el derribo y la unión de ARN en células de mamíferos.
  • Evaluar la eficacia y la especificidad de LwaCas13a para la orientación del ARN.
  • Desarrollar una plataforma programable para el estudio del ARN en células vivas.

Principales métodos:

  • Se examinaron 15 ortólogos de Cas13a para identificar la variante más efectiva (LwaCas13a).
  • Se expresa heterológicamente en las células de mamíferos y plantas.
  • Utilizó LwaCas13a catalíticamente inactivo para la unión y el seguimiento del ARN.

Principales resultados:

  • LwaCas13a demostró una eliminación eficaz de las transcripciones reporteras y endógenas.
  • Se alcanzan niveles de eliminación comparables a la interferencia de ARN pero con una especificidad mejorada.
  • Se ha demostrado el seguimiento programable de transcripciones en células vivas utilizando LwaCas13a inactivo.

Conclusiones:

  • CRISPR-Cas13a es una plataforma versátil para la investigación de ARN en células de mamíferos.
  • LwaCas13a ofrece una herramienta específica y programable para el knockdown y la visualización de ARN.
  • Esta tecnología tiene aplicaciones potenciales en el desarrollo terapéutico para enfermedades relacionadas con el ARN.