Poster abstracts

Poster number 126 submitted by Mohammed Enamul Hoque

Transcription regulation by CRISPR/dCas9 complex in the context of G-quadruplex structure

Mohammed Enamul Hoque (Department of Chemistry and Biochemistry, Kent State University), Hamza Balci (Department of Physics, Kent State University), Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University)

CRISPR-Cas is an RNA-mediated adaptive immune system found in bacteria and archaea that protects host cells from foreign DNA invasion. CRISPR-Cas9 is guided by a single guide RNA (sgRNA), which hybridizes with complementary genomic sequences and induces a double-stranded break (DSB). The use of an engineered nuclease-deficient Cas9, known as dCas9, allows the system to be repurposed for targeting genomic DNA without cleaving it. According to recent studies, dCas9 is a versatile, RNA-guided DNA recognition platform that enables precise, scalable, and robust RNA-guided transcription regulation. There is a prevalence of guanosine-rich sequences in the human genome and transcriptome and such sequences have a propensity to adopt secondary structures called G-quadruplexes (GQ). Despite abundant information and practical knowledge about the CRISPR-Cas9 technology, its capabilities and limitations in targeting DNA GQ structures have not yet been systematically investigated. To address this knowledge gap, we utilized CRISPR/dCas9 system for targeting in the vicinity of GQ forming sequence of human tyrosine hydroxylase (TH) gene promoter and discovered that TH mRNA expression levels could both be up or down regulated. This interesting observation still needs a mechanistic explanation which would require an understanding of how the interactions between dCas9 and RNA polymerase (RNAP) is affected by the neighboring GQ structures. When GQ was present in the non-template strand, only a full-length RNA band was observed. However, when GQ was present in the template strand, we observed some truncated RNA bands in addition to the full-length RNA. The truncated RNA products could be due to the GQ-mediated stalls on the RNAP progression. Then, we targeted the same GQ containing sequence by using CRISPR/dCas9 complex in four different sites of the PQS, two in the template and the remaining two in the non-template strand. When target site was on the non-template strand of T7 RNAP, we found that dCas9 served as the dominant block compared to the GQ stall. Interestingly, when target site was on the template strand, GQ served as the dominant block, with no dCas9 blockade. Furthermore, smFRET experiment will be performed to gain the mechanistic and dynamic understanding of the interactions among RNAP, dCas9 and GQ.

Keywords: Transcription Regulation, CRISPR-dCas9, G-quadruplex