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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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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
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Estructura y ingeniería del complejo de efectores CRISPR-Cas7-11 tipo III-E

Kazuki Kato1, Wenyuan Zhou2, Sae Okazaki1

  • 1Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.

Cell
|June 1, 2022
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Resumen

El sistema Cas7-11 CRISPR-Cas ofrece capacidades duales de orientación de ARN para células bacterianas y de mamíferos. Las ideas estructurales permitieron diseñar una variante compacta de Cas7-11 para aplicaciones de eliminación de ARN in vivo utilizando vectores AAV.

Palabras clave:
CRISPR y sus derivadosCasos 7 y 11Orientación al ARNCrio-EM

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Área de la Ciencia:

  • Biología molecular
  • Biología estructural
  • Tecnologías de edición de genes

Sus antecedentes:

  • El efector CRISPR-Cas tipo III-E, Cas7-11, posee actividades duales de RNase únicas.
  • Estas actividades incluyen el procesamiento de ARN precursor CRISPR (pre-crRNA) y la escisión de ARN objetivo guiado por ARN CRISPR (crRNA).
  • Cas7-11 representa una nueva plataforma para la orientación del ARN tanto en los sistemas bacterianos como en los mamíferos.

Objetivo del estudio:

  • Determinar la estructura de alta resolución del complejo Cas7-11 unido al ARNcr y al ARN objetivo.
  • Para aclarar los mecanismos moleculares subyacentes a las actividades duales de la RNasa de Cas7-11.
  • Diseñar una variante compacta del Cas7-11 para aplicaciones in vivo.

Principales métodos:

  • Microscopía cryoelectrónica (cryo-EM) con una resolución de 2,5 Å.
  • Pruebas bioquímicas para confirmar la actividad catalítica.
  • La ingeniería racional de las proteínas de Cas7-11.

Principales resultados:

  • El estudio reveló la arquitectura modular de Cas7-11, que comprende siete dominios y cuatro enlaces.
  • El análisis estructural identificó las funciones de dominios específicos en el procesamiento de crRNA y el reconocimiento de ARN objetivo.
  • Datos bioquímicos correlacionados con los hallazgos estructurales relativos al posicionamiento de los residuos catalíticos para la escisión del ARN.
  • Una variante compacta Cas7-11 (Cas7-11S) fue diseñada con éxito.

Conclusiones:

  • La estructura determinada proporciona información crítica sobre el mecanismo de acción de Cas7-11 para la orientación del ARN.
  • El Cas7-11S diseñado facilita el empaquetado AAV de un solo vector para la eliminación eficiente de la transcripción en las células humanas.
  • Este trabajo permite futuras aplicaciones in vivo del sistema Cas7-11 para la modulación del ARN.