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Related Concept Videos

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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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.
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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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Genome expansion by a CRISPR trimmer-integrase.

Joy Y Wang1,2, Owen T Tuck1,2, Petr Skopintsev2,3,4

  • 1Department of Chemistry, University of California, Berkeley, CA, USA.

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|June 14, 2023
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CRISPR-Cas systems use a novel trimmer-integrase enzyme, fusing Cas1-Cas2 with an exonuclease, to accurately capture and integrate foreign DNA. This mechanism ensures self DNA is protected, preventing autoimmune reactions in microorganisms lacking Cas4.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • CRISPR-Cas systems provide adaptive immunity in microorganisms by integrating foreign DNA fragments.
  • Distinguishing self from non-self DNA is crucial for CRISPR function and preventing autoimmunity.
  • While Cas4 assists in some CRISPR adaptation, many systems lack this protein.

Purpose of the Study:

  • To investigate the mechanism of DNA integration in CRISPR-Cas systems that lack the Cas4 protein.
  • To elucidate the role of an alternative pathway involving a DnaQ-like exonuclease in CRISPR adaptation.
  • To characterize the structure and function of the Cas1-Cas2/exonuclease fusion enzyme.

Main Methods:

  • Cryo-electron microscopy to visualize the CRISPR trimmer-integrase complex.
  • Biochemical assays to study DNA processing and integration.
  • Structural analysis of the enzyme before and during DNA integration.

Main Results:

  • A type I-E CRISPR system utilizes a fused Cas1-Cas2/exonuclease (trimmer-integrase) for DNA integration.
  • The exonuclease (DEDDh) selects and processes DNA using the protospacer adjacent motif (PAM).
  • Structural data reveals asymmetric DNA processing, generating defined, PAM-containing substrates and protecting self DNA.

Conclusions:

  • CRISPR systems lacking Cas4 employ fused or recruited exonucleases for the faithful acquisition of new immune sequences.
  • The trimmer-integrase mechanism ensures accurate DNA integration and prevents self-targeting.
  • This study reveals an elegant alternative pathway for CRISPR adaptation and genome integrity maintenance.