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The CRISPR-Cas system is an adaptive immune defense system for bacteria and archaea against exogenous nucleic acid invasion using Cas effector proteins and guided by gRNA. Currently, CRISPR technology has been widely used as a genome editing tool in biology, medicine, agriculture and other fields. In addition, the discovery of some Cas enzymes with "incidental cleavage" activity suggests that CRISPR systems can be used to detect critical analytes such as nucleic acids, proteins, small molecules and metal ions.
The Chemical Fundamentals of CRISPR-associated Enzymes (Xie, S.Y. et al, Analytica Chimica Acta, 2021)
When the target nucleic acid is present in the system to be detected, the Cas protein specifically recognizes the target sequence under the guidance of gRNA and forms a complex, which is then stimulated with "trans cleavage activity" against the cleaved single-stranded nucleic acid, shredding the fluorescent labeled probe in the system and emitting a fluorescent signal to complete the detection process, and the presence or absence of the target nucleic acid is determined by whether the system is luminescent.
For the detection of non-nucleic acid targets, the non-nucleic acid signal can be converted into a nucleic acid signal that can be detected using a nucleic acid detection strategy by using a conversion element such as a functional nucleic acid.
In Vitro Detection Procedures for Nucleic Acids (Xie, S.Y. et al, Analytica Chimica Acta, 2021)
High Specificity
Single-base resolution detection of biosensors is fully possible.
High Sensitivity
Target analytes can be detected at concentrations as low as 10-18 mol/L.
High Flexibility
Only the given gRNA sequence needs to be changed for different gene targets.
The CRISPR-Cas9 system uses DNA as the recognition target and exerts the cleavage of Cas9 effector under the guidance of gRNA, and this type of assay has high specificity and sensitivity to the substrate being detected. And it shows strong DNA virus detection in clinical specimens with high detection sensitivity.
This is achieved by subjecting dsDNA fragments treated with phosphatase to Cas9-mediated specific cleavage of the target sequence. In contrast to the uncut product, the cleaved DNA ends are ligated to the adapter. The cleaved product is amplified with adaptor-specific PCR primers and the target sequence is identified by sequencing.
The CRISPR-associated protein Cas12a, an RNA-guided endonuclease in the second type V CRISPR system, has incidental cleavage activity, also contains a RuvC domain, and cleaves 18-25 nt of target DNA downstream of the PAM (TTTN) sequence under crRNA guidance.
The principle of Cas12 for molecular detection is that Casl2a targets dsDNA under the mediation of crRNA, forming a ternary complex, which activates the non-specific ssDNA trans-cleavage activity. The Cas12-based assay developed with this property enables sensitive, specific, and rapid detection of SNP typing, DNA viruses, and RNA viruses.
CRISPR-Cas13 is an RNA-inducible RNA targeting system that specifically cleaves single-stranded target RNA under the guidance of gRNA, and continues to maintain activity after cleavage and cuts other non-target RNA, which is the "collateral cleavage" capability. Based on this feature, CRISPR-Cas13 can detect multiple targets, such as Zika virus, Dengue virus, pathogenic bacterial strains and their drug-resistance genes, SNP typing, and ctDNA.
Utilizing the DeteCas™ platform based on the CRISPR-Cas system, CD BioSciences can provide various services, such as analyte detection service and detection method development service. If you are interested in these services, please contact us.
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