TY - CHAP
T1 - Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network
AU - Okusha, Yuka
AU - Eguchi, Takanori
N1 - Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - CCN2/CTGF
KW - CRISPR/Cas9
KW - cellular communication network
KW - exosomes
KW - genetic knockout
KW - genome editing
KW - moonlighting/matrix metalloproteinase
KW - tissue microenvironment
UR - http://www.scopus.com/inward/record.url?scp=85141719886&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85141719886&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-2744-0_11
DO - 10.1007/978-1-0716-2744-0_11
M3 - Chapter
C2 - 36370349
AN - SCOPUS:85141719886
T3 - Methods in Molecular Biology
SP - 157
EP - 167
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -