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Bases estructurales para la degradación del ADN guiada por ARN por cascada y Cas3

Yibei Xiao¹, Min Luo², Adam E Dolan¹

¹Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.

Science (New York, N.Y.)

|June 9, 2018

Resumen

El sistema tipo I-E CRISPR-Cas utiliza Cascade y Cas3 para degradar el ADN. Las estructuras Cryo-EM revelan cómo Cas3 se dirige y degrada el ADN después de que Cascade identifique objetivos, asegurando una escisión precisa del ADN.

Área de la Ciencia:

Biología molecular
La genética
La bioquímica

Sus antecedentes:

Los sistemas CRISPR-Cas de tipo I proporcionan inmunidad adaptativa en las bacterias.
Involucran a Cascade para el objetivo del ADN y a Cas3 para la degradación.
Comprender el mecanismo es crucial para las aplicaciones de ingeniería genómica.

Objetivo del estudio:

Para aclarar la base estructural de la degradación del ADN por el tipo I-E sistema CRISPR-Cas.
Para visualizar la interacción entre Cascade, R-loop y Cas3.
Para entender los pasos secuenciales de búsqueda y degradación del ADN objetivo.

Principales métodos:

Microscopía cryoelectrónica (cryo-EM) con una resolución de 3,7 y 4,7 angstroms.
Análisis estructural del complejo Cascade/R-loop/Cas3.
Pruebas bioquímicas para estudiar la degradación del ADN.

Principales resultados:

Determinación de la estructura del complejo tipo I-E de cascada/bucle R/Cas3 preparado para la degradación del ADN.
El Cas3 mostrado distingue conformaciones de cascada específicas para evitar la escisión fuera del objetivo.
Se visualizó el reclutamiento y el nicking de la hebra no objetivo (NTS) por el dominio de la nucleasa de Cas3.
Capturado el estado post-nicking, revelando la retracción NTS para la degradación mediada por Cas3.

Conclusiones:

El estudio proporciona información estructural clave sobre el mecanismo de degradación del ADN guiado por ARN por sistemas tipo I-E CRISPR-Cas.
La especificidad de Cas3 está mediada por el reconocimiento de las conformaciones en cascada.
Los hallazgos iluminan los procesos secuenciales de búsqueda, cortado y degradación del ADN.

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