Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network

Yuka Okusya, Takanori Eguchi

Research output: Chapter in Book/Report/Conference proceedingChapter


The Cellular Communication Network Factor (CCN) family is composed of six members: CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3. The second member, CCN2/CTGF is a matricellular protein that promotes extracellular matrix (ECM) synthesis and controls angiogenesis. On the other hand, moonlighting/matrix metalloproteinase 3 (MMP3) is an ECM-degrading enzyme that also functions as an intracellular transcription factor. Importantly, extracellular MMP3 is uptaken into cells, translocating into nuclei, and transcriptionally activating CCN2/CTGF gene in cancer and chondrocytes. Thus, the MMP3-CTGF axis balances the matrix metabolism and turnover in the tissue and tumor microenvironments. We established an MMP3 knockout cell line using the CRISPR/Cas9 system, demonstrating the sequential regulatory events of the MMP3-CCN2 axis in the microenvironment. Notably, our protocol is useful for generation of CCN knockout cells as well. Here we serve a protocol of the CRISPR/Cas9-based gene targeting in cultured cells for investigating cellular communication network.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages11
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • cellular communication network
  • CRISPR/Cas9
  • exosomes
  • genetic knockout
  • genome editing
  • moonlighting/matrix metalloproteinase
  • tissue microenvironment

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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