TY - JOUR
T1 - Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus
AU - Xiong, Xiong
AU - Tasan, Ipek
AU - Yang, Che
AU - Zhang, Meng
AU - Hernandez Gonzalez, Gabriela A.
AU - Liu, Shuting
AU - Chaturvedi, Pankaj
AU - Belmont, Andrew S.
AU - Zhao, Huimin
N1 - Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/5/19
Y1 - 2023/5/19
N2 - Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.
AB - Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.
KW - CRISPR-Sirius
KW - DNA replication
KW - HSPA1 gene
KW - TAL effector
KW - Tet operator/Tet repressor
KW - imaging
KW - site-specific targeting
UR - http://www.scopus.com/inward/record.url?scp=85154059749&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85154059749&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.2c00433
DO - 10.1021/acssynbio.2c00433
M3 - Article
C2 - 37058298
AN - SCOPUS:85154059749
SN - 2161-5063
VL - 12
SP - 1424
EP - 1436
JO - ACS synthetic biology
JF - ACS synthetic biology
IS - 5
ER -