Recently, the research group led by Associate Professor Liu Yizhen from the College of Chemistry and Environmental Engineering has made significant breakthroughs in the field of CRISPR molecular diagnostic technology, providing key technical solutions for its industrial application. Under the guidance of Associate Professor Liu Yizhen, Master's student Zhong Jialing (Class of 2022) has successively published two research papers on CRISPR diagnostics in Analytical Chemistry, a Nature Index journal.

1. First Achievement: Titled "A Single-Tube, Single-Enzyme CRISPR System (UNISON) with Internal Controls for Accurate Nucleic Acid Detection," this paper was published in Analytical Chemistry (Nature Index, CAS Class I Top Journal). Zhong Jialing, a 2022 master's student in chemistry, serves as the first author, while Associate Researcher Chen Yong and Associate Professor Liu Yizhen are the corresponding authors. The College of Chemistry and Environmental Engineering at Shenzhen University is the sole affiliated institution.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins have been widely used in molecular diagnostics. However, CRISPR diagnostic systems, which primarily rely on the rising rate of fluorescence signals to indicate target nucleic acid concentrations, are susceptible to systematic errors caused by various factors such as enzyme activity, reaction conditions, and instrument performance. Establishing internal controls is crucial for enhancing the accuracy, reliability, and commercial viability of CRISPR systems. Unlike qPCR, which supports dual fluorescence channels for internal control, the nonspecific trans-cleavage activity of Cas proteins has hindered the implementation of internal controls. In this study, the authors developed a single-tube, single-enzyme CRISPR nucleic acid detection system with internal controls (UNISON) for accurate nucleic acid detection. By extending crRNA and modifying it with different fluorophores and quenchers, they enabled specific targets to exclusively cleave corresponding folded crRNAs, generating distinct fluorescence signals. This design integrates internal control functionality into the CRISPR/Cas system, achieving accurate and reliable detection of clinical hepatitis B virus samples. This research holds significant implications for constructing stable and reliable CRISPR diagnostic reagent systems.

Paper Link：https://pubs.acs.org/doi/10.1021/acs.analchem.4c03403

2. Second Achievement: Titled "SPARC: An Orthogonal Cas12a/Cas13a Dual-Channel CRISPR Platform for Reliable SNV Identification and Mutation Confirmation," this paper was also published in Analytical Chemistry. Zhong Jialing, under the guidance of Associate Professor Liu Yizhen, serves as the first author, with Associate Professor Liu Yizhen as the corresponding author. The College of Chemistry and Environmental Engineering at Shenzhen University remains the sole affiliated institution.

Rapid and reliable detection of single nucleotide variants (SNVs) is essential for precise pathogen diagnostics, genetic mutation screening, and personalized medicine. Existing CRISPR-based nucleic acid detection platforms often suffer from ambiguous signal interpretation, limited specificity, and complex assay workflows. In this work, the research team introduced SPARC (Specific and Precise Mutation Recognition with Cas12a/Cas13a), a novel orthogonal dual-channel CRISPR assay that significantly enhances the reliability of SNV detection. SPARC integrates Acidaminococcus sp. Cas12a (AsCas12a), which detects a conserved region as an internal reference, and the recently identified DNA-activated Leptotrichia buccalis Cas13a (LbuCas13a), which exhibits exceptional intrinsic SNV specificity without requiring engineered crRNA mismatches. This orthogonal design uniquely resolves the diagnostic ambiguity between genuine SNVs and target absence. Combined with recombinase polymerase amplification (RPA) and T7 exonuclease digestion, the SPARC platform achieved a sensitivity as low as 1 aM. The team demonstrated the platform's robust clinical applicability by successfully detecting and differentiating hepatitis B virus (HBV) and clinically significant YMDD resistance mutations. This study presents an innovative and versatile CRISPR-based solution with substantial potential for advancing clinical diagnostics and precision medicine.

Paper Link：https://pubs.acs.org/doi/10.1021/acs.analchem.5c02141