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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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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CRISPR01:59

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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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Homologous Recombination02:31

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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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CRISPR and crRNAs02:53

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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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RNA Editing02:23

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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Edición de ARN con CRISPR-Cas13

David B T Cox1,2,3,4,5,6, Jonathan S Gootenberg1,2,3,4,7, Omar O Abudayyeh1,2,3,4,6

  • 1Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA.

Science (New York, N.Y.)
|October 27, 2017
PubMed
Resumen
Este resumen es generado por máquina.

Los investigadores desarrollaron una nueva plataforma de edición de ARN llamada REPAIR, utilizando CRISPR-Cas13 y ADAR2. Este sistema corrige con precisión las mutaciones causantes de enfermedades a nivel de ARN en las células de mamíferos, ofreciendo un potencial terapéutico.

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Área de la Ciencia:

  • Biotecnología
  • Biología molecular
  • Ingeniería genética

Sus antecedentes:

  • La edición de ARN ofrece una estrategia prometedora para el tratamiento de enfermedades genéticas mediante la corrección de secuencias relacionadas con la enfermedad.
  • Los sistemas CRISPR-Cas de tipo VI, específicamente la enzima Cas13, son nucleasas guiadas por ARN con potencial para la manipulación de ARN programable.

Objetivo del estudio:

  • Para diseñar un ortólogo Cas13 para una robusta destrucción de ARN.
  • Demostrar la edición de ARN en células de mamíferos utilizando Cas13 catalíticamente inactivo (dCas13) para guiar a la adenosina desaminasa que actúa sobre el ARN 2 (ADAR2).
  • Desarrollar una plataforma versátil de edición de ARN para la investigación y la terapia.

Principales métodos:

  • Perfilamiento de los sistemas CRISPR-Cas tipo VI.
  • Ingeniería de una variante catalíticamente inactiva de Cas13 (dCas13).
  • Coexpresión de dCas13 con la adenosina desaminasa que actúa sobre el ARN 2 (ADAR2) para la edición dirigida de ARN de adenosina a inosina (A a I).
  • Desarrollo de una variante de alta especificidad y un sistema minimizado para la entrega viral.

Principales resultados:

  • Se ha demostrado el éxito de la edición de ARN en células de mamíferos utilizando el sistema REPAIR.
  • Mostró la capacidad de editar transcripciones completas con mutaciones patógenas sin restricciones de secuencia estrictas.
  • Diseñado una variante de alta especificidad y un sistema compacto adecuado para la entrega viral.

Conclusiones:

  • El sistema REPAIR representa una nueva y programable plataforma de edición de ARN.
  • Esta tecnología tiene una amplia aplicabilidad en investigación básica, desarrollo terapéutico para enfermedades genéticas y biotecnología.
  • REPAIR ofrece un enfoque flexible para corregir las secuencias de ARN, evitando la necesidad de modificar el ADN.