CRISPR/Cas Systems towards Next-Generation Biosensing

doi:10.1016/j.tibtech.2018.12.005

Trends in Biotechnology

Volume 37, Issue 7, July 2019, Pages 730-743

https://doi.org/10.1016/j.tibtech.2018.12.005 Get rights and content

Highlights

CRISPR/Cas biosensing systems transfer the sequence information of target nucleic acids to detectable signals such as fluorescence and colorimetric values.

CRISPR/Cas biosensing systems are versatile platforms for nucleic acid detection that can be used for pathogen detection and genotyping, cancer mutation detection, and single nucleotide polymorphism (SNP) identification.

The biosensing methods employing these Cas effectors rely on the collateral cleavage activities of Cas13 and Cas12.

CRISPR/Cas biosensing allows highly sensitive, specific, rapid, cost-efficient, and multiplex detection of target nucleic acids, and support point-of-care use without the need for technical expertise and complicated equipment.

Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms.

Section snippets

CRISPR/Cas System Coming into Biosensing Applications

Molecular diagnostics is critical for life sciences, biosecurity, food safety, and environmental monitoring 1, 2. Detection of nucleic acids is a major molecular diagnostic practice that has been continuously growing in the past few decades. Besides various PCR-based nucleic acid detection methods, different isothermal amplification and nucleic acid hybridization methods have also been established 3, 4. However, most of the existing techniques have trade-offs in performance metrics such as

Classification of CRISPR/Cas Biosensing Systems

In a CRISPR/Cas system, a pre-CRISPR RNA (crRNA) is transcribed from the CRISPR array and further processed to yield the mature crRNA, which serves as the guider to navigate the Cas effectors. The Cas effectors, which can be formed by either a single protein or a complex of proteins, are the enzymatic units possessing the target-dependent cleavage activity. Among the established CRISPR/Cas-based nucleic acid biosensing systems, the fundamental difference remains in implementing different Cas

Pros and Cons of Different CRISPR/Cas Biosensing Systems

Most of these introduced CRISPR/Cas biosensing systems have advantages of simplicity to develop/re-develop, ultra-high resolution to single-base variation, at least fM or mostly aM concentration sensitivity, and no need for dedicated instruments. Additionally, these nucleic acid biosensing systems have the potential to satisfy various needs of POC and field deployment diagnostics, as they can be modified to work on a variety of in vitro mediums efficiently and robustly. Their tolerance to

Exploration of CRISPR/Cas for Additional Biosensing Purposes

The mechanism of dCas9-based genome imaging is similar to that of dCas9-based biosensing, but with a different purpose. When dCas9 is fused with a fluorescent protein, the fusion protein (e.g., dCas9-EGFP) can specifically bind to a target sequence in living cells, allowing dynamical observation of the specific loci [41]. To achieve high signal output, the fusion protein is often targeted to highly repetitive sequences such as telomeres and centrosomes, recruiting multiple copies of the

Concluding Remarks and Future Perspectives

Based on current understanding, the entirety of the discovered CRISPR/Cas family has been categorized into two classes with six subtypes 52, 53. Cas effectors from class 2 are commonly composed of only one protein to fulfil both the recognition and target cleavage functions. The simplicity and high efficiency have promised wide applications of Class 2 Cas effectors, including type II (i.e., Cas 9), type V (i.e., Cas12a and Cas12b), and type VI (i.e., Cas13a and Cas13b) effectors, not only in

Acknowledgments

This work was financially supported by funding from the Australia Research Council Future Fellowship (FT160100039), the ARC Centre of Excellence for Nanoscale BioPhotonics CE140100003, and the National Natural Science Foundation of China (Grant 21575045). Yi Li would like to thank the University of New South Wales for providing PhD Scholarship.

Glossary

CRISPR/Cas system: a type of unique genomic element originally discovered in bacteria and archaea, serving as an adaptive immune system to defend the invasion of phage or other foreign nucleic acids. The system comprises a short repeated DNA array called the clustered regularly interspaced short palindromic repeats (CRISPR) and a sort of CRISPR-associated proteins (Cas) expressed by cas genes.
CRISPR diagnostics: an approach to molecular diagnostics that employs Cas effectors. For example,

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Trends in Biotechnology

ReviewCRISPR/Cas Systems towards Next-Generation Biosensing

Highlights

Section snippets

CRISPR/Cas System Coming into Biosensing Applications

Classification of CRISPR/Cas Biosensing Systems

Pros and Cons of Different CRISPR/Cas Biosensing Systems

Exploration of CRISPR/Cas for Additional Biosensing Purposes

Concluding Remarks and Future Perspectives

Acknowledgments

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Review
CRISPR/Cas Systems towards Next-Generation Biosensing