Friday, May 3, 2024
5/3/2024
Project Summary: Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) proteins, particularly Cas9, have provided unprecedented programmable control on targeting specific sequences. Cas9 and its engineered mutant endonuclease-dead Cas9 (dCas9), use CRISPR-RNA (crRNA) as a guide to target a 20-nucleotide long DNA sequence. An active CRISPR complex requires near-perfect complementarity and R-loop formation between crRNA and the target DNA. Despite its frequent use in different applications and extensive knowledge about it, the capabilities and limitations of Cas9 for targeting sequences that are prone to folding into secondary structures are not known. Secondary structures, such as G-quadruplexes (GQs), are abundant in the human genome and are physiologically and medically significant. In two recent publications, we demonstrated examples of how GQs could inhibit target recognition and R-loop formation, distort the structure, and inhibit Cas9-mediated DNA cleavage. We propose to perform systematic single molecule and bulk biophysical studies to determine how GQs located in the vicinity of Cas9 target site impact critical aspects of its function. Targeting potentially GQ forming sequences (PQS) also provides new application venues for CRISPR and could solve a long-standing problem in achieving transient and sequence-specific transcription regulation through PQS. PQS have been identified in promoters of prominent oncogenes and transcription factors that are upregulated in many cancers. Stabilizing GQs in promoters of these oncogenes with small molecules has resulted in suppression of their transcription, which made this approach a potential anti- cancer therapy. However, despite their structural specificity, these small molecules are not sequence specific. Site-directed mutagenesis has also been used to modify PQS to control gene expression; however, such changes are permanent. Targeting PQS with sequence-specificity and modulating gene expression temporarily have not been possible with alternative approaches, which we propose can be achieved with dCas9. We hypothesize that gene expression can be up or down regulated by targeting PQS or their vicinity with dCas9. We present data on tyrosine hydroxylase (TH) transcription which supports this hypothesis. The proposal has two specific aims: (1) To determine how GQs in target strand or non-target strand of DNA influence target recognition, binding, conformational states, kinetics, and cleavage activities of Cas9 (and dCas9) using in vitro single molecule fluorescence, particularly FRET, and ensemble methods. (2) To target PQS in promoters of c-MYC, KRAS, and TH genes by dCas9 to determine whether their mRNA and protein expressions can be modulated in relevant cell lines. While c-MYC codes for a transcription factor that is up-regulated in many cancers, KRAS is an oncogene that is significant in signaling and cell proliferation. TH is the rate limiting enzyme in dopamine biosynthesis.
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