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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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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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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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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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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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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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A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.

Shrutee Jakhanwal1,2,3, Brady F Cress1,2,3, Pascal Maguin4

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

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CRISPR-Cas9 protein precisely trims DNA to 30 base pairs for integration into bacterial genomes. This Cas9 function is key for generating bacterial immunological memory, independent of guide RNA sequence.

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Area of Science:

  • Molecular Biology
  • Bacterial Immunity
  • Genome Engineering

Background:

  • CRISPR-Cas9 systems provide adaptive immunity in bacteria by integrating foreign DNA fragments into the CRISPR locus via the Cas1-Cas2 integrase.
  • The mechanism by which specific DNA lengths are generated for this integration process remains largely unknown.
  • Understanding DNA processing is crucial for elucidating type II-A CRISPR immunity.

Purpose of the Study:

  • To investigate the mechanism of DNA trimming during CRISPR-mediated bacterial immunity.
  • To identify the molecular players and catalytic activities responsible for generating specific DNA lengths for integration.
  • To explore the role of Cas9 in DNA processing within the CRISPR-Cas system.

Main Methods:

  • Biochemical assays using purified CRISPR-Cas proteins (Cas1, Cas2, Csn2, Cas9) and guide RNA.
  • Analysis of DNA products generated by the integrase supercomplex.
  • Characterization of the catalytic activity of the Cas9 HNH active site in DNA trimming.

Main Results:

  • A supercomplex of guide RNA, Cas1, Cas2, Csn2, and Cas9 generates precisely 30-base pair DNA molecules.
  • The Cas9 HNH active site is responsible for the exonucleolytic trimming of DNA.
  • This DNA trimming activity is independent of the guide RNA sequence.

Conclusions:

  • Cas9 possesses a previously unrecognized catalytic function in DNA processing.
  • This function enables the generation of specific DNA lengths required for bacterial genome integration and immunological memory.
  • The findings reveal a novel role for Cas9 in prokaryotic adaptive immunity.