CRISPR-based diagnostic methods

Overview

Many CRISPR enzymes can be useful in diagnosis of infectious diseases (viruses, bacteria, etc.), as well as cancer and many other diseases that in some way involve a nucleic acid sequence (either RNA or DNA).

CRISPR diagnostic methods

CRISPR systems are usually used for genome editing, but they can also be used in applications that have no genome editing component. One of the most important new applications of CRISPR is in diagnosis and detection of infectious diseases, using enzymes in the Cas9, Cas12, Cas13, and Cas14 families. Many of the methods are still in the research phase and have not entered widespread clinical use, but some have been authorized by governmental regulatory bodies for specific clinical uses.

Cas9 methods for disease detection can be divided into those that use enzymatically active Cas9 [1] and those that use catalytically inactive (dead) Cas9, known as dCas9 [2, 3]. All the Cas9-based methods of disease detection can theoretically detect any DNA sequence having a Cas9 target site and PAM sequence. Another way Cas9 can be used in disease detection is by cleavage of a pathogen’s genomic DNA into pieces sized appropriately for next generation sequencing [4].

Cas12-, Cas13-, and Cas14-based methods of disease detection are rapidly growing in popularity. SHERLOCK™ (Specific High-sensitivity Enzymatic Reporter unLOCKing, Sherlock Biosciences) and DETECTR™ (DNA endonuclease-targeted CRISPR trans reporter, Mammoth Biosciences, Inc.) are the best known of these methods [5, 6]. These technologies depend on collateral cleavage, a process in which Cas enzymes cut not only the target DNA or RNA but also a large number of unrelated DNA or RNA molecules nearby in solution. Because labeled RNA or DNA can be added to the assay, this process allows for significant signal amplification.

In addition to infectious diseases, inherited diseases and cancer can also be detected with CRISPR-based methods. Indeed, any disease that involves any DNA or RNA sequence can potentially be detected by CRISPR-based means. However, development of CRISPR-based assays for non-infectious diseases is not as advanced compared with development of such assays for infectious diseases, so more research is needed in this area.


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References

1. Pardee K, Green AA, et al. (2016) Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell 165(5):1255–1266.

2. Zhang Y, Qian L, et al. (2017) Paired design of dCas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains.  ACS Synth Biol 6(2):211–216.

3. Guk K, Keem JO, et al. (2017) A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex. Biosens Bioelectron 95:67–71.

4. Quan J, Langelier C, et al. (2019) FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences. Nucleic Acids Res 47(14):83.

5. Gootenberg JS, Abudayyeh OO, et al. (2018) Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Biosens Bioelectron Science 360(6387):439–444.

6. Chen JS, Ma E, et al. (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360(6387):436–439.

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