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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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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-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
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Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network.

Yuka Okusha1, Takanori Eguchi2

  • 1Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|November 12, 2022
PubMed
Summary

Matrix metalloproteinase 3 (MMP3) activates CCN2/CTGF gene expression, regulating tissue and tumor microenvironments. This study details a CRISPR/Cas9 protocol for investigating this cellular communication axis.

Keywords:
CCN2/CTGFCRISPR/Cas9cellular communication networkexosomesgenetic knockoutgenome editingmoonlighting/matrix metalloproteinasetissue microenvironment

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Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The Cellular Communication Network Factor (CCN) family comprises six proteins involved in various biological processes.
  • CCN2/CTGF, a matricellular protein, promotes extracellular matrix (ECM) synthesis and angiogenesis.
  • Matrix metalloproteinase 3 (MMP3) is an ECM-degrading enzyme with a dual role as an intracellular transcription factor.

Purpose of the Study:

  • To investigate the regulatory mechanism of the MMP3-CCN2 axis in cellular communication.
  • To establish a protocol for generating knockout cell lines using CRISPR/Cas9 for studying this axis.
  • To elucidate the sequential regulatory events of MMP3-CCN2 in microenvironments.

Main Methods:

  • CRISPR/Cas9 gene targeting system for creating knockout cell lines.
  • Cultured cell models to study cellular communication networks.
  • Analysis of gene expression and protein interactions within cellular microenvironments.

Main Results:

  • Demonstrated that extracellular MMP3 is internalized and translocates to the nucleus.
  • Showed MMP3 transcriptionally activates the CCN2/CTGF gene in cancer and chondrocytes.
  • Established an MMP3 knockout cell line to study the MMP3-CCN2 axis.
  • Developed a protocol applicable for generating CCN knockout cells.

Conclusions:

  • The MMP3-CCN2 axis plays a critical role in balancing matrix metabolism and turnover in tissue and tumor microenvironments.
  • The established CRISPR/Cas9 protocol provides a valuable tool for investigating cellular communication networks and generating knockout cell lines.
  • This research offers insights into the molecular mechanisms governing cell-matrix interactions and gene regulation.