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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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Mismatch Repair01:20

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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
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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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CRISPR and crRNAs02:53

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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.
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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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The Antiviral System of Bacteria and Archaea: CRISPR01:23

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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...
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Bases estructurales para la vigilancia de las discrepancias mediante CRISPR-Cas9

Jack P K Bravo1, Mu-Sen Liu1, Grace N Hibshman1,2

  • 1Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.

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La edición del genoma CRISPR-Cas9 está limitada por la escisión del ADN fuera del objetivo. Este estudio revela cómo Cas9 reconoce desajustes, lo que permite el diseño de variantes de alta fidelidad con una mayor precisión y eficiencia de escisión en el objetivo.

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Área de la Ciencia:

  • Biología molecular
  • La genética
  • La bioquímica

Sus antecedentes:

  • La edición del genoma CRISPR-Cas9 es una herramienta poderosa, pero sufre de escisión del ADN fuera del objetivo debido a la mala comprensión de los mecanismos de reconocimiento de desajustes.
  • Las variantes existentes de Cas9 con una mejor discriminación de desajuste a menudo exhiben tasas reducidas de escisión en el objetivo, lo que limita su potencial terapéutico.

Objetivo del estudio:

  • Aclarar los mecanismos estructurales subyacentes al reconocimiento y la escisión del desajuste de Cas9.
  • Para diseñar la próxima generación de variantes de alta fidelidad Cas9 con mayor especificidad y actividad retenida.

Principales métodos:

  • Se empleó microscopía crioelectrónica guiada por cinética (cryo-EM) para capturar las estructuras de Cas9 en varias etapas de la escisión del desajuste del ADN.
  • Se utilizó la mutagénesis dirigida al sitio para alterar los residuos específicos involucrados en la estabilización del desajuste.

Principales resultados:

  • Se observó una conformación dúplex guía lineal distinta de ARN-ADN, que inhibe la activación de Cas9, en presencia de desajustes.
  • Los desajustes distales al motivo adyacente del protospacer son estabilizados por un bucle de dominio RuvC reorganizado.
  • La mutagenesis de los residuos estabilizadores de desajuste redujo con éxito la escisión fuera del objetivo mientras se conservaba la escisión rápida en el objetivo.

Conclusiones:

  • Dirigirse a las regiones Cas9 involucradas en la tolerancia al desajuste ofrece una estrategia viable para desarrollar herramientas de edición de genoma de alta fidelidad.
  • Esta investigación proporciona una base estructural para el diseño de sistemas CRISPR-Cas9 mejorados con mayor especificidad para aplicaciones terapéuticas.