Journal
ACS SYNTHETIC BIOLOGY
Volume 10, Issue 5, Pages 972-978Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00067
Keywords
CRISPR; Cas9; dCas9; G-quadruplex; transcription regulation; DNA cleavage
Categories
Funding
- NIH [1R15GM123443]
- University Research Council
- Graduate Student Senate at Kent State University
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Using dCas9, transcription levels of TH can be modulated by targeting different parts of a G-rich sequence within the promoter. The presence of GQs in target sequences impacts DNA cleavage activity of Cas9, with a significant reduction in cleavage activity when targeting high-stability GQs. Additionally, this reduction is more pronounced for the G-rich strand compared to the complementary C-rich strand.
Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.
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