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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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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CRISPR01:59

CRISPR

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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...
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Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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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...
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Caspases01:24

Caspases

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Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside...
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Video Experimental Relacionado

Updated: Aug 23, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

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Bases estructurales para la actividad fuera del objetivo de Cas9

Martin Pacesa1, Chun-Han Lin2, Antoine Cléry3

  • 1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Cell
|October 28, 2022
PubMed
Resumen

La edición del genoma CRISPR-Cas9 puede dirigirse a secuencias de ADN no deseadas, lo que plantea preocupaciones de seguridad. El análisis estructural revela que el emparejamiento de bases no canónico y la acomodación de las deleciones permiten la unión fuera del objetivo, lo que guía el diseño mejorado del ARN guía.

Palabras clave:
CRISPR y sus derivadosEn el caso deCristalografía de rayos Xemparejamiento de basesEdición del genomaARN guíadesajusteLa nucleasafuera del objetivo

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Área de la Ciencia:

  • Biología molecular
  • La genética
  • La bioquímica

Sus antecedentes:

  • La nucleasa Cas9 asociada a CRISPR es una poderosa herramienta de edición del genoma.
  • La especificidad de Cas9 se basa en la complementariedad del ARN guía con el ADN objetivo.
  • La escisión fuera del objetivo por Cas9 plantea riesgos de seguridad para aplicaciones clínicas.

Objetivo del estudio:

  • Para aclarar la base estructural de la unión y la escisión fuera del objetivo de Cas9.
  • Para entender cómo Cas9 acomoda los desajustes y las deleciones en el ADN objetivo.
  • Informar el diseño racional de sistemas de edición de genoma más seguros basados en Cas9.

Principales métodos:

  • Cristalografía de rayos X de los complejos de ADN Cas9.
  • Análisis de sustratos fuera del objetivo con complementariedad variable.
  • Comparación estructural de los modos de unión en el objetivo y fuera del objetivo.

Principales resultados:

  • Cas9 se une al ADN fuera del objetivo a través de interacciones de emparejamiento de bases no canónicas.
  • Las deleciones de un solo nucleótido se acomodan con saltos de bases o múltiples pares no canónicos.
  • Los desajustes PAM-distales inducen desparejamiento dúplex y cambios conformacionales en Cas9.

Conclusiones:

  • Las ideas estructurales explican la actividad fuera del objetivo de Cas9.
  • Los hallazgos facilitan un diseño mejorado de ARN guía para una mayor especificidad.
  • Este trabajo ayuda a desarrollar mejores algoritmos de predicción fuera del objetivo para la tecnología CRISPR.