Novel strategy proposed for selectively targeting g-quadruplex at specific genome loci

DNA G-quadruplexes (G4s) are quadruple helix structures formed by a continuous guanine-rich DNA sequence. Although DNA G4s are thought to be involved in various biological processes, in many cases their causative effects are largely unclear due to a lack of suitable strategies for selectively stabilizing DNA G4s at specific loci. 

In a study published in Nature Cell Biology on July 3, Prof. QU Xiaogang from the Changchun Institute of Applied Chemistry (CIAC) of the Chinese Academy of Sciences and his colleagues presented a novel strategy for achieving DNA G4 stabilization at specific genome loci by combining CRISPR and G4-stabilizing proteins or compounds.  

The researchers employed clustered regularly interspaced short palindromic repeat-associated dead Cas9 (CRISPR/dCas9) targeting constructs to endow G4-stabilizing molecules with selectivity among different G4s. In addition, the dCas9/G4-stabilizing molecule complexes could stabilize any G4 structure in the genome by strategic single-guide RNA (sgRNA) design without a significant effect on other non-targeted G4 in live cells.  

The researchers discovered that fusion of the G4-stabilizing protein nucleolin with dCas9 could specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Igta as well as telomere G4s, thus leading to cell proliferation arrest, inhibition of myoblast differentiation, and cell senescence, respectively. 

In addition, they discovered that CRISPR could confer intra-G4 selectivity to the G4-binding compounds pyridodicarboxamide (PDC) and pyridostatin (PDS). This strategy was used to stabilize the G4s within the promoter regions of two well-established lncRNAs, NEAT1 and MALAT1, as well as several key genes implicated in ferroptosis, oxidative stress, hypoxia response, and the MAPK pathway. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated PDC and PDS enabled a more precise investigation into the biological functionality of de novo G4s. 

This strategy could pave the way for illustrating the association of specific G4s with defined biological processes or human diseases. In addition, it may open a new avenue in live cells for screening G4 probes or G4 drug candidates, by specifically targeting only the G4 of interest with a low risk of off-target effects, thus promoting G4-based disease diagnosis and therapy.