Poster abstracts
Poster number 32 submitted by Mohammad Lutful Kabir
Combined CRISPR-activation and CRISPR-interference capabilities using dCas and G-quadruplex Structures
Mohammad Lutful Kabir (Chemistry and Biochemistry,Kent State University ), Sineth G. Kodikara (Physics,Kent State University), Hamza Balci (Physics,Kent State University)
Abstract:
We demonstrate that both CRISPR interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex forming sequence (PQS) in the c-Myc promoter and VEGF UTR IRES A with nuclease-dead Cas9 (dCas9) and dca13d. The achieved suppression and activation in Burkitt's Lymphoma and HUVEC cell line, and in in vitro studies, are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the G-quadruplex was destabilized and c-Myc mRNA and protein levels increased by 2.1-fold and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the non-template strand with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8-fold and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. In the case of VEGF A protein level, we found more than 5-fold decrease in this protein compared to the induced hypoxia condition. We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription and translation is modulated via the interactions of RNA polymerase, CRISPR-dCas9,dcas13d and the G-quadruplex.
References:
Mohammad Lutful Kabir, Sineth G Kodikara, Mohammed Enamul Hoque, Sajad Shiekh, Janan Alfehaid, Soumitra Basu, Hamza Balci, Combining CRISPR activation and interference capabilities using dCas9 and G-quadruplex structures, NAR Molecular Medicine, Volume 2, Issue 1, January 2025, ugaf001, https://doi.org/10.1093/narmme/ugaf001
Keywords: CRISPR, dCas, G-quadruplex