Poster abstracts
Poster number 75 submitted by Mohammad Lutful Kabir
Combined CRISPR-activation and CRISPR-interference capabilities using dCas9 and G-quadruplex Structures
Mohammad Lutful Kabir (Chemistry and Biochemistry,Kent State University ), Sineth G. Kodikara (Physics,Kent State University), Mohammed Enamul Hoque (Chemistry and Biochemistry,Kent State University ), Soumitra Basu (Chemistry and Biochemistry,Kent State University ), Hamza Balci (Physics,Kent State University)
Abstract:
Catalytically dead Cas9 (dCas9) has been employed in CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPRa) to modulate transcription (gene expression). Altering the stability of G-quadruplex structures located in promoters is also known to modulate transcription (gene expression). We demonstrate that both CRISPRi and CRISPRa are feasible within the same system by targeting the vicinity of a putative GQ sequence in c-Myc promoter with CRISPR-dCas9. The achieved suppression and activation levels are at or beyond above those reported with alternative approaches, such as drug like small molecules which lack sequence specificity or site-directed mutations which are permanent. In addition to demonstrating up or down regulation of c-Myc in Burkitt's Lymphoma cell line (at both RNA and protein levels) and the resulting impact on cell viability, we demonstrate extensive in vitro studies probing the underlying mechanism. Our studies illustrate dCas9 to be more likely to block RNAP progression and suppress transcription when the non-template strand is targeted, the orientation in which the GQ is also stabilized. Targeting multiple sites in the non-template strand simultaneously with dCas9 further suppressed transcription (~2-fold for single site and ~5-fold for dual site targeting). When the template strand was targeted, CRISPR-dCas9 destabilized the GQ and enhanced transcription (~2-fold). Our study demonstrates the promising potential of targeting PQS with CRISPR-dCas9 as a means to differentially regulate transcription in a simple system that combines both CRISPRa and CRISPRi capabilities.
Keywords: CRISPR,dCas9,G-quadruplex, Transcription Regulation, , RNA Polymerase