Abstract
The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications.