CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

Polymerase chain reaction (PCR)‐based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR‐mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point‐of‐care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR‐based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics‐target preamplification‐free detection, microRNA (miRNA) testing, non‐nucleic‐acid detection, and SARS‐CoV‐2 detection‐are also covered.

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR‐based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification‐free nucleic acid detection via harnessing the trans‐cleavage mechanism of Cas13a and ultrasensitive graphene field‐effect transistors (gFETs). CRISPR Cas13a‐gFET achieves the detection of SARS‐CoV‐2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS‐CoV‐2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a‐gFET among the most sensitive amplification‐free nucleic acid diagnostic platforms to date.

Back Cover: Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors (Angew. Chem. Int. Ed. 32/2022)

Nucleic acid detection plays a critical role in medical diagnostics, environmental monitoring, and food safety. In their Research Article (e202203826), Xue Gao, Yi Zhang and co‐workers developed a new biosensor for amplification‐free nucleic acid detection via harnessing the trans‐cleavage mechanism of Cas13a and ultrasensitive graphene field‐effect transistors (gFETs). The illustration shows the Cas13a‐mediated RNA trans‐cleavage on a gFET surface for sensor signal transduction.

Rücktitelbild: Amplification‐Free Detection of SARS‐CoV‐2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field‐Effect Transistors

Der Nachweis von Nukleinsäuren spielt eine wichtige Rolle in der medizinischen Diagnostik, der Umweltüberwachung und der Lebensmittelsicherheit. In ihrem Forschungsartikel (e202203826) entwickelten Xue Gao, Yi Zhang und Mitarbeiter einen neuen Biosensor für den amplifikationsfreien Nukleinsäurenachweis, indem sie den trans‐Spaltungsmechanismus von Cas13a und ultrasensitive Graphen‐Feldeffekttransistoren (gFETs) nutzten. Die Abbildung zeigt die Cas13a‐vermittelte RNA‐trans‐Spaltung auf der gFET‐Oberfläche für die Sensorsignalübertragung.

Amplification-Free Detection of SARS-CoV-2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field-Effect Transistors

Der Nachweis von Nukleinsäuren spielt eine wichtige Rolle in der medizinischen Diagnostik, der Umweltüberwachung und der Lebensmittelsicherheit. In ihrem Forschungsartikel (DOI: 10.1002/ange.202203826) entwickelten Xue Gao, Yi Zhang und Mitarbeiter einen neuen Biosensor für den amplifikationsfreien Nukleinsäurenachweis, indem sie den trans-Spaltungsmechanismus von Cas13a und ultrasensitive Graphen-Feldeffekttransistoren (gFETs) nutzten. Die Abbildung zeigt die Cas13a-vermittelte RNA-trans-Spaltung auf der gFET-Oberfläche für die Sensorsignalübertragung.