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Una plataforma de alto rendimiento para identificar inhibidores de moléculas pequeñas de CRISPR-Cas9

Basudeb Maji¹, Soumyashree A Gangopadhyay¹, Miseon Lee²

¹Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.

|May 4, 2019

Resumen

Los científicos desarrollaron las primeras moléculas pequeñas sintéticas para controlar la edición de genes CRISPR-Cas9. Estos inhibidores celulares permeables ofrecen un control preciso y reversible sobre las tecnologías de ingeniería genómica.

Área de la Ciencia:

Biología molecular
La bioquímica
Biología Química

Sus antecedentes:

El control preciso de CRISPR-Cas9 es crucial para el avance de la ingeniería genómica.
Los métodos existentes para controlar la actividad de CRISPR-Cas9 tienen limitaciones.
El desarrollo de inhibidores de moléculas pequeñas ofrece un nuevo enfoque para regular CRISPR-Cas9.

Objetivo del estudio:

Desarrollar una plataforma generalizable para la identificación de inhibidores sintéticos de pequeñas moléculas de Streptococcus pyogenes Cas9 (SpCas9).
Para crear inhibidores celulares permeables, reversibles y estables para el control preciso de la actividad de SpCas9.
Establecer un método para la identificación rápida de inhibidores de SpCas9 y nucleasas relacionadas.

Principales métodos:

Desarrolló ensayos de alto rendimiento para las funciones de SpCas9, incluida la unión de motivos adyacentes al protospacer.
Examinó una colección diversa de pequeñas moléculas parecidas a productos naturales.
Se utilizaron relaciones estructura-actividad para identificar un farmacóforo para la inhibición de SpCas9.

Principales resultados:

Se identificaron los primeros inhibidores sintéticos de molécula pequeña de SpCas9 (<500 Da) que son permeables a las células, reversibles y estables.
Control demostrado de la dosis y el tiempo de SpCas9 y tecnologías de SpCas9 catalizadas, incluida la activación de la transcripción.
Se estableció un farmacóforo para la inhibición de SpCas9.

Conclusiones:

Se ha establecido una plataforma generalizable para el descubrimiento de inhibidores sintéticos, en miniatura, permeables a las células y reversibles de SpCas9.
Estas pequeñas moléculas proporcionan un control preciso temporal y dependiente de la dosis sobre la actividad de CRISPR-Cas9.
La plataforma puede ampliarse para identificar inhibidores de las nucleasas asociadas a CRISPR de próxima generación.

Palabras clave:

CRISPR-Cas9 también S. pyogenes Cas9 Edición de la base dCas9 y Edición del genoma cribado de alto rendimiento Inhibidor de moléculas pequeñas Anti-CRISPR sintéticos y sus derivados Activación de la transcripción

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