CRISPR/Cas Biosensing System: Opportunities and Challenges

Rather than being famous in gene editing field only, by revealing the collateral cleavage activity of Cas12a, Cas13a, Cas14 effectors, and more, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) (i.e., CRISPR/Cas) systems has recently received significant credits in the field of diagnostics with the capability of developing novel CRISPR/Cas medicated biosensors with superior sensitivity and specificity. Exciting development of CRISPR/Cas based biosensors has thrived quickly in recent 2-3 years, and a variety of different biosensing systems have now been developed successfully such as SHERLOCK, SHERLOCKv2, DETECTR, HOLMES, etc. Despite the features of high sensitivity and specificity, all the known CRISPR/Cas involved biosensing systems are specialized to detect nucleic acids. Recent studies have demonstrated that CRISPR/Cas systems are providing excellent biosensing capability for detection of non-nucleic acids, such as small molecules, proteins, exosomes, metal ions, et al. Although the most popular signal read-out in CRISPR/Cas biosensors is fluorescence, recently, various signal read-out modalities such as colorimetric, electrochemiluminescence, electrochemical and electrical have been applied in CRISPR/Cas biosensing systems. Furthermore, they can be multiplexed, enabling a single diagnostic test to identify the presence of multiple targets. Coupling of the CRISPR/Cas detection with lateral flow device-based signal readout, will allow for the highly sensitive and accurate in-field detection of pathogens and viruses.

Preamplification techniques such as rolling circle amplification (RCA), recombinase polymerase amplification (RPA), polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), etc. have significantly boosted the detection sensitivity of CRISPR/Cas. However, they inevitably overshadow Cas effectors and neglect the intrinsic detection capability of Cas effectors due to the exponential format, nonspecific background products that lead to false-positive results. Therefore, research focusing on preamplification based CRISPR/Cas systems is active. Additionally, application of CRISPR/Cas biosensing systems for multiplexed detection and in vivo biosensing is also the spotlight.   The CRISPR/Cas biosensing technology has massive potential in facilitating the development of modern analytical science, and will only be advanced as analytical chemists, engineers and molecular biologists continue to improve collaborative capability.

Learning Objectives:

1. Define the key factors affect the efficiency of Cas12a autocatalysis reaction.

2. Discuss the advantages and disadvantages of the Cas12a autocatalysis reaction developed here.

3. Evaluate the biosensing performance of AutoCAR for nucleic acid detection.