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Nucleasa CRISPR-Cas9 diseñada con espacio de orientación expandido

Hiroshi Nishimasu1, Xi Shi2,3, Soh Ishiguro4,5,6

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.

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Los científicos diseñaron una variante de la enzima Cas9 (SpCas9-NG) para reconocer motivos adyacentes de protospacer NG relajados (PAM). Esto expande las capacidades de edición del genoma al apuntar a los loci genómicos previamente inaccesibles en las células humanas.

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Área de la Ciencia:

  • Biología molecular
  • La genómica
  • La bioquímica

Sus antecedentes:

  • La enzima Cas9 es una herramienta clave en la edición del genoma, que permite la escisión del ADN dirigido.
  • La enzima Streptococcus pyogenes Cas9 (SpCas9) comúnmente utilizada está limitada por su requisito para un motivo adyacente de protospacer NGG específico (PAM).
  • Esta especificidad NGG PAM restringe el rango de sitios genómicos que pueden ser objeto de edición.

Objetivo del estudio:

  • Para diseñar una variante SpCas9 con capacidades de reconocimiento PAM relajadas.
  • Para superar las limitaciones impuestas por el estricto requisito NGG PAM del tipo salvaje SpCas9.
  • Ampliar el alcance de los loci genómicos seleccionables para la edición del genoma basada en CRISPR.

Principales métodos:

  • Ingeniería racional de la enzima SpCas9 para crear una variante (SpCas9-NG) con especificidad PAM alterada.
  • Cristalografía de rayos X para determinar la base estructural del reconocimiento de NG PAM por SpCas9-NG.
  • Ensayos funcionales en células humanas para evaluar la capacidad de SpCas9-NG para inducir indels en los sitios de NG PAM.
  • Fusión de SpCas9-NG con desaminasa citidina inducida por activación (AID) para probar las capacidades de edición de bases en los sitios NG PAM.

Principales resultados:

  • Se ha diseñado con éxito una variante SpCas9-NG capaz de reconocer NG PAM relajados.
  • La estructura cristalina reveló que SpCas9-NG acomoda NG PAM a través de interacciones específicas de base alteradas.
  • SpCas9-NG demostró eficacia en la inducción de inserciones/deleciones (indels) en sitios endógenos de NG PAM en células humanas.
  • SpCas9-NG fusionado con AID facilitó las conversiones dirigidas de C a T en los sitios de NG PAM en células humanas.

Conclusiones:

  • SpCas9-NG representa un avance significativo en la tecnología de edición del genoma basada en Cas9.
  • El SpCas9-NG diseñado amplía el rango de orientación, permitiendo la edición en loci genómicos previamente inaccesibles.
  • La fusión SpCas9-NG con AID ofrece una herramienta versátil para la edición precisa de bases en los sitios NG PAM, mejorando las aplicaciones de ingeniería genómica.