CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives

Abstract

CRISPR/Cas system is becoming an increasingly influential technology that has been repositioned in nucleic acid detection. A preamplification step is usually required to improve the sensitivity of CRISPR/Cas-based detection. The striking biological features of CRISPR/ Cas, including programmability, high sensitivity and sequence specificity, and single-base resolution. More strikingly, the target-activated trans-cleavage could act as a biocatalytic signal transductor and amplifier, thereby empowering it to potentially perform nucleic acid detection without a preamplification step. The reports of such work are on the rise, which is not only scientifically significant but also promising for futuristic end-user applications. This review started with the introduction of the detection methods of nucleic acids and the CRISPR/ Cas-based diagnostics (CRISPR-Dx). Next, we objectively discussed the pros and cons of preamplification steps for CRISPR-Dx. We then illustrated and highlighted the recently developed strategies for CRISPR/Cas-powered amplification-free detection that can be realized through the uses of ultralocalized reactors, cascade reactions, ultrasensitive detection systems, or others. Lastly, the challenges and futuristic perspectives were proposed. It can be expected that this work not only makes the researchers better understand the current strategies for this emerging field, but also provides insight for designing novel CRISPR-Dx without a preamplification step to win practicable use in the near future.

A Thermostable Cas12b-Powered Bioassay Coupled with Loop-Mediated Isothermal Amplification in a Customized “One-Pot” Vessel for Visual, Rapid, Sensitive, and On-Site Detection of Genetically Modified Crops

“…According to the number of Cas effectors in the step of interfering exogenous nucleic acids, CRISPR/Cas can be divided into two types. The first type of Cas effectors includes type I, type III, and type IV, which are characterized by utilizing a variety of different protein modules together and forming protein/nucleic acid complexes to realize their functions, accounting for about 90% of the loci of the CRISPR/Cas system. , The second type almost exists in bacteria, including type II (Cas9), type V (Cas12), and type VI (Cas13), accounting for the remaining 10%, among which Cas12, Cas13, and Cas14 have not only cis -cleavage activity but also trans -cleavage activity. , While single guide RNA (sgRNA) recognizes a complementary target, it activates nuclease activity and cleaves the target sequence specifically, which is called cis -cleavage activity . The activated Cas effector can cleave the responsive single-stranded DNA (ssDNA) probe arbitrarily to generate fluorescence signals, which are called trans -cleavage activity.…”

Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene Microsphere Reporter System

“…CRISPR/Cas capability of target-activated trans -cleavage to function as a biocatalytic signal transducer and amplifier is particularly impressive . This allows CRISPR/Cas systems to target nucleic acids without the requirement for amplification while also providing automation, downsizing, customization, deployment, and simplicity.…”