TY - CHAP
T1 - Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network
AU - Okusya, Yuka
AU - Eguchi, Takanori
N1 - Funding Information:
T.E was supported by JSPS Kakenhi, grant numbers 17K11642-TE, 20K09904-CS, 19H03817-MT, 20H03888-HN, 20K20611-MT, 20H03888-HN, 21H03119-TY, and 21K08902-HY. Y.O was supported by JSPS overseas research fellowship. The authors thank Eriko Aoyama, Satoshi Kubota, Kuniaki Okamoto, Chiharu Sogawa, Eman Taha, Masaharu Takigawa, and Manh Tien Tran for useful information, discussion, materials, or experimentation.
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 - cellular communication network
KW - CRISPR/Cas9
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 -
