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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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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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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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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/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
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Estructuras del complejo de integración del genoma CRISPR

Addison V Wright1, Jun-Jie Liu1,2, Gavin J Knott1

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

Science (New York, N.Y.)
|July 22, 2017
PubMed
Resumen

La integrasa Cas1-Cas2 utiliza la estructura del ADN, no solo la secuencia, para integrar el ADN extraño en los loci de CRISPR. Este mecanismo, ayudado por IHF, asegura una inmunidad adaptativa bacteriana precisa contra los virus.

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Área de la Ciencia:

  • Biología molecular
  • Microbiología
  • Biología estructural

Sus antecedentes:

  • Los sistemas CRISPR-Cas proporcionan inmunidad adaptativa en las bacterias contra los fagos.
  • La integrasa Cas1-Cas2 es esencial para la adquisición de nuevos espaciadores y la integración de ADN extraño en el locus CRISPR.

Objetivo del estudio:

  • Elucidar los mecanismos estructurales de la integración del ADN por la integrasa Cas1-Cas2.
  • Comprender cómo se logra la selección del sitio de integración durante la expansión de la matriz CRISPR.

Principales métodos:

  • Cristalografía de rayos X para determinar las estructuras de los complejos Cas1-Cas2-ADN.
  • Microscopía criolectrónica para el complejo de integración de locus CRISPR completo con IHF.

Principales resultados:

  • Las estructuras cristalinas revelan Cas1-Cas2 unido al donante y al ADN objetivo en estados intermedios y productos.
  • La estructura cryo-EM muestra el complejo de integración incluyendo el factor de acogida de integración (IHF).
  • La selección del sitio de integración está dictada por el reconocimiento indirecto de la secuencia de ADN y la deformación del ADN dependiente de la secuencia.

Conclusiones:

  • Cas1-Cas2 utiliza las características estructurales del ADN, favoreciendo la deformación de la secuencia de repetición y flanqueo, para la integración específica del sitio.
  • La unión de IHF induce una fuerte flexión del ADN, mejorando la especificidad y la eficiencia de la integración al poner un motivo aguas arriba en contacto con Cas1.
  • Estos hallazgos explican el mecanismo de reconocimiento de la estructura de ADN dependiente de la secuencia por Cas1-Cas2 para la expansión de la matriz CRISPR selectiva del sitio en la inmunidad adaptativa bacteriana.