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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

2.6K
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...
2.6K
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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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...
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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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Video Experimental Relacionado

Updated: Mar 29, 2026

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
11:37

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

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Nucleasas Cas9 de ingeniería racional con especificidad mejorada

Ian M Slaymaker1, Linyi Gao2, Bernd Zetsche1

  • 1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Science (New York, N.Y.)
|December 3, 2015
PubMed
Resumen
Este resumen es generado por máquina.

Las variantes de Cas9 diseñadas, llamadas Cas9 de especificidad mejorada (eSpCas9), reducen significativamente la escisión no intencional del ADN en la edición del genoma. Estas herramientas mejoradas de Cas9 mantienen una orientación precisa para aplicaciones de edición de genes más seguras.

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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Last Updated: Mar 29, 2026

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Área de la Ciencia:

  • Biología molecular
  • Biotecnología
  • La genómica

Sus antecedentes:

  • La endonucleasa guiada por ARN Cas9 es una herramienta clave para la edición del genoma, que permite modificaciones específicas del ADN.
  • Las enzimas Cas9 crean rupturas de doble cadena (DSB) en ubicaciones genómicas específicas guiadas por moléculas de ARN.
  • Una limitación significativa de Cas9 es su potencial para dividir sitios fuera del objetivo, comprometiendo la precisión y seguridad de la edición.

Objetivo del estudio:

  • Diseñar variantes de Cas9 (eSpCas9) de mayor especificidad derivadas de Streptococcus pyogenes Cas9 (SpCas9).
  • Evaluar rigurosamente la especificidad y la actividad de escisión en el objetivo de las variantes de eSpCas9 en células humanas.
  • Para abordar el desafío de la escisión fuera del objetivo en las aplicaciones de edición del genoma.

Principales métodos:

  • La ingeniería de proteínas guiada por la estructura se empleó para modificar SpCas9.
  • Se utilizó la secuenciación profunda dirigida para detectar la escisión de ADN mediada por Cas9 en posibles sitios fuera del objetivo.
  • Se realizó un análisis imparcial de todo el genoma fuera del objetivo para evaluar exhaustivamente la actividad de Cas9 en todo el genoma.

Principales resultados:

  • Las variantes eSpCas9 diseñadas demostraron una reducción significativa en la escisión del ADN fuera del objetivo en comparación con el SpCas9 de tipo salvaje.
  • Las variantes de eSpCas9 mantuvieron una alta eficiencia de escisión en el objetivo, asegurando una edición precisa en los lugares deseados.
  • Tanto los análisis dirigidos como los de genoma completo confirmaron la mayor especificidad de las variantes de eSpCas9.

Conclusiones:

  • Las variantes SpCas9 de especificidad mejorada (eSpCas9) minimizan efectivamente los efectos fuera del objetivo en la edición del genoma.
  • eSpCas9 conserva una robusta actividad de escisión en el objetivo, lo que lo convierte en una herramienta confiable para la edición genética precisa.
  • Estas variantes de Cas9 diseñadas ofrecen una mayor seguridad y eficacia para diversas aplicaciones de edición del genoma que requieren una alta especificidad.