Combining the CRISPR Activation and Interference Capabilities Using dCas9 and G-Quadruplex Structures.

Area of Science:

• Molecular Biology
• Gene Regulation
• Biotechnology

Background:

• The c-Myc oncogene plays a critical role in cell proliferation and is frequently dysregulated in cancer.
• G-quadruplex forming sequences (PQS) in promoter regions are emerging as key regulatory elements.
• CRISPR-based tools offer precise genome editing capabilities.

Purpose of the Study:

• To investigate the efficacy of targeting a PQS in the c-Myc promoter using nuclease-dead Cas9 (dCas9) for gene regulation.
• To demonstrate both CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) at the c-Myc locus.
• To elucidate the mechanistic details of dCas9 interaction with PQS and its impact on transcription.

Main Methods:

• Utilized dCas9 to target the vicinity of a PQS in the c-Myc promoter in a Burkitt's Lymphoma cell line.
• Employed CRISPR interference by targeting the non-template strand and CRISPR activation by targeting the template strand.
• Conducted in vitro biophysical studies to complement cellular assays and understand transcription modulation.

Main Results:

• Targeting the template strand with dCas9 destabilized the G-quadruplex, increasing c-Myc mRNA and protein by 2.1-fold and 1.6-fold, respectively.
• Targeting the non-template strand with dCas9 reduced c-Myc mRNA and protein by 1.8-fold and 2.5-fold, with dual-site targeting achieving 3.6-fold and 9.8-fold reductions.
• Cell viability assays showed corresponding reductions with non-template strand targeting, indicating functional consequences.

Conclusions:

• CRISPR-dCas9 targeting of a c-Myc promoter PQS enables robust gene activation and repression.
• The study provides a novel strategy for modulating oncogene expression via G-quadruplex manipulation.
• Findings are supported by in vitro biophysical data, offering mechanistic insights into transcription control.