A Single-Tube, Single-Enzyme Clustered Regularly Interspaced Short Palindromic Repeats System (UNISON) with Internal Controls for Accurate Nucleic Acid Detection

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been widely applied in molecular diagnostics. Unlike the Ct value quantification method of PCR, the CRISPR system mainly relies on the rise of the rate of the fluorescence signal to indicate the concentration of the target nucleic acid, which is susceptible to system errors caused by various factors, such as reaction conditions and instrument performance. Therefore, establishing internal controls is essential to improve the accuracy, reliability, and commercial feasibility of the CRISPR system. However, the nonspecific trans-cleavage activity of Cas proteins presents a challenge in establishing internal controls. In this study, we developed unified nucleic acid detection with a single-tube, one-enzyme system (UNISON) for accurate nucleic acid detection with internal controls. By extending the crRNA and modifying it with different fluorophores and quenchers, we achieved that the specific target can only specifically cleave the corresponding folded crRNA and generate a corresponding fluorescence signal. With this design, we established an internal control, achieving accurate and reliable detection of clinical samples of the hepatitis B virus. Integrating internal controls into the CRISPR/Cas system demonstrates significant potential in medical diagnostics and virus monitoring.