The development of innovative systems to analyze genetic variants is key to unlocking the mysteries of congenital disorders. A recent study introduces dual-enSERT, a groundbreaking dual-color fluorescent reporter system utilizing the CRISPR/Cas9 technology. This system allows researchers to rapidly and quantitatively compare the activities of human enhancer variants within live mice—achieving results within less than two weeks.
Enhancers are regulatory DNA sequences found within non-coding regions of the genome. They play significant roles by modulating the transcription of genes, with their activity being highly dependent on cell-type specificity. Importantly, nearly 90% of disease risk-associated genetic variations occur within non-coding regions, yet the functional impacts of most enhancer variants remain largely unexplored. The introduction of the dual-enSERT system marks a significant advancement, allowing for simultaneous assessment of enhancer allele activities and addressing previous limitations faced by researchers.
The dual-enSERT system enables the integration of enhancer variants driving distinct fluorescent proteins (such as eGFP and mCherry) within the same mouse genome, providing direct visualization of enhancer activities. Through testing 15 previously uncharacterized non-coding variants linked to neurodevelopmental disorders—including autism spectrum disorder and congenital limb malformations—researchers have effectively demonstrated how enhancer activity can be altered by single nucleotide polymorphisms.
For example, the study effectively linked the human reference ZRS allele to its enhancer activity compared with the pathogenic 404G>A variant responsible for pre-axial polydactyly. Observed results indicated significant increases (up to 31-fold) of fluorescence from the enhancer variant, confirming its dysfunction via ectopic gene expression. Such findings highlight the potential of dual-enSERT as not merely diagnostic but also as prospectively therapeutic by identifying specific targets for gene regulation.
Adding to the dual-color capability, coupling dual-enSERT with single-cell transcriptomics provided insights at cellular resolution. It uncovered pathways activated by enhancer misregulation when specific enhancer variants lead to genetic disorders. By executing differential gene analyses, researchers found similarities between normally active and ectopically expressed genes, allowing the characterization of genetic pathways linked to abnormal gene regulation and potential targets for rehabilitation.
The research concluded by emphasizing the dual-enSERT technology’s role as it progresses from genetic association to functional validation of enhancer variants. Not only does it provide efficient screening of both rare and common genetic variants, but it also accelerates enhancer-variant function studies across numerous congenital disorders.
Overall, this promising technology sets the stage for future investigations and advancements, propelling the current genetic and medical fields toward enhanced functionality and mechanistic understandings of human disease.