Video Experimental Relacionado

Updated: Jan 13, 2026

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

Diseño computacional completo de Cas9 de Staphylococcus aureus con PAM relajado y capacidad de direccionamiento

Youcai Xiong¹, Li-Kuang Tsai¹, Jun Zhou¹

¹Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI 48109, USA.

bioRxiv : the preprint server for biology

|January 7, 2026

Resumen

El diseño computacional creó KRH, una variante de Cas9 de Staphylococcus aureus, que amplía los sitios genómicos dirigibles al relajar los requisitos del motivo adyacente al protoespaciador. Esto acelera las tecnologías de edición genética de próxima generación.

Área de la Ciencia:

Genética y Genómica
Ingeniería de Proteínas
Bioinformática

Sus antecedentes:

La edición genética CRISPR-Cas9 es potente pero limitada por el reconocimiento del motivo adyacente al protoespaciador (PAM).
La Cas9 de Staphylococcus aureus (SaCas9) es adecuada para la administración in vivo pero tiene un PAM NNGRRT restrictivo.

Objetivo del estudio:

Desarrollar una variante de SaCas9 con especificidad PAM relajada utilizando diseño computacional.
Evaluar la eficiencia de edición genómica y de bases de la variante diseñada.

Principales métodos:

Se utilizó UniDesign, un flujo de trabajo de diseño de proteínas computacional, para crear variantes de SaCas9.
Se introdujeron mutaciones puntuales (E782K/N968R/R1015H) para generar la variante KRH.
Se evaluaron las eficiencias de edición en múltiples tipos de células humanas.

Principales resultados:

La variante KRH reconoce eficientemente un PAM NNNRRT ampliado.
KRH demostró eficiencias de edición genómica y de bases comparables a la variante KKH.
El análisis estructural reveló que KRH ajusta las interacciones para relajar la especificidad del PAM.

Conclusiones:

El diseño computacional puede diseñar eficazmente variantes de Cas9 con especificidad PAM alterada.
La variante KRH ofrece una herramienta valiosa para expandir las aplicaciones de edición genómica.
Este enfoque proporciona una estrategia escalable para desarrollar nuevas nucleasas Cas9.

Palabras clave:

edición genómica CRISPR-Cas9 Staphylococcus aureus Cas9 diseño computacional ingeniería de proteínas especificidad PAM variante KRH herramientas de edición genética

Más Videos Relacionados

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Published on: February 26, 2019

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

Videos de Conceptos Relacionados

CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

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...

CRISPR

CRISPR

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...

CRISPR and crRNAs

CRISPR and crRNAs

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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

The Antiviral System of Bacteria and Archaea: CRISPR

The Antiviral System of Bacteria and Archaea: CRISPR

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...