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Related Concept Videos

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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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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Homologous Recombination02:31

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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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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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Related Experiment Video

Updated: Jun 6, 2025

Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Substrate Generation for Endonucleases of CRISPR/Cas Systems

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CRISPR/Cas Enzyme Catalysis in Liquid-Liquid Phase-Separated Systems.

Yaqin Zhang1,2, Jianai Chen2, Zhina Wu3

  • 1Department of Clinical Pharmacy, The First Hospital of Jilin University, Jilin University, Changchun, Jilin, 130021, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|November 22, 2024
PubMed
Summary

Investigating CRISPR/Cas systems in simulated intracellular environments revealed altered enzyme activity. Cis-cleavage by Cas12a was enhanced, while trans-cleavage was suppressed, offering insights into gene editing tools.

Keywords:
ATPSCRISPRCas12aCas13a

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Bioengineering

Background:

  • The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) system, a bacterial immune mechanism, is a vital tool in bioengineering.
  • Understanding CRISPR/Cas catalysis within intracellular environments is crucial for advancing CRISPR-based technologies.

Purpose of the Study:

  • To investigate the catalytic mechanisms of CRISPR/Cas systems in a simulated intracellular environment.
  • To elucidate the behavior of Cas12a and Cas13a activities under physiological conditions.

Main Methods:

  • Utilized an aqueous two-phase system (ATPS) composed of PEG and dextran to mimic intracellular conditions.
  • Analyzed the distribution of nucleic acids and proteins within the ATPS.
  • Assessed the cis- and trans-cleavage activities of Cas12a and validated findings with Cas13a.

Main Results:

  • Nucleic acids and proteins preferentially partitioned into the dextran-rich phase of the ATPS.
  • Cas12a exhibited enhanced cis-cleavage activity and suppressed trans-cleavage activity in the ATPS.
  • This modulation of activity was confirmed for Cas13a and was not due to reporter diffusion limitations.

Conclusions:

  • The study provides novel insights into CRISPR/Cas catalytic mechanisms under simulated physiological conditions.
  • Findings explain the controlled activity of Cas12a and Cas13a within intracellular compartments.
  • This research may contribute to the development of improved CRISPR-based molecular tools.