CRISPR-Cas Systems for Detection of Nucleic Acids: PCR Empire now has a Vigorous Rival!

Editorial: In 2020, Jennifer Doudna and Emmanuelle Charpentier were awarded Nobel Prize in chemistry for genome editing using the CRISPR-CAS system. Clustered Regular Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) evolved as an adaptive immune system for prokaryotes against viruses and foreign nucleic acids. In 2012 Doudna, Charpentier, and colleagues reported that the CRISPR-Cas system could be harnessed as a cut and paste programmable tool for genome editing (1). Up to date, this system has revolutionized the field of genetic manipulation. Recently, novel applications for this system, such as nucleic acid detection, have been introduced. Quantitative PCR is the gold standard for nucleic acid detection and quantification but has disadvantages such as demand for equipment and reagents, trained personnel, and time (2). CRISPR-Cas systems can provide fast, reliable, and highly accurate detection of DNA and RNA in diverse fields such as ecological studies, molecular research, and clinical identification of pathogens. CRISPR-Cas-based detection of nucleic acids is established using versions of Cas protein such as Cas13a and Cas12a instead of Cas9. These Cas proteins have an incredible ability called collateral activity (non-specific trans-cleavage of bystander quenched-fluorescent reporter RNA/DNA molecules upon activation through complementary matching of crRNA and RNA/DNA of interest). This Quenched-fluorescent Reporter molecule emits light after cleaving by Cas proteins, and this light can be measured and quantified as a criterion for detecting and quantifying nucleic acid of interest.

Specific high sensitivity enzymatic reporter unlocking (SHERLOCK) and DNA endonuclease targeted CRISPR trans reporter (DETECTR) are two main methods that have been used for the CRISPR-Cas detection of nucleic acids. SHERLOCK uses Cas13 (an endo-ribonuclease) and detects single-stranded RNA (it can also detect dsDNA by using RPA & T7 RNA-polymerase in the pre-amplification phase) (3). DETECTR uses Cas12a (an endo-deoxyribonuclease) and detects single and double-stranded DNA (4). Both approaches can be done after isothermal amplification of the initial specimen with Recombinase Polymerase Amplification (RPA).

Although CRISPR-Cas detection of nucleic acids has not been approved for diagnosis yet, promising pre-clinical research results were obtained. Doudna et al. demonstrated that DETECTR could diagnose human papillomavirus in the clinical specimen and discriminate between two similar subtypes HPV16 and HPV18 (5). In another study, Lee et al. Used SHERLOCK for ultrasensitive malaria diagnosis in symptomatic and asymptomatic patients. Their results demonstrated that SHERLOCK could detect the Plasmodium parasite and differentiate between four Plasmodium species causing malaria in about one hour (6). 

Conclusion: These new methods of nucleic acid detection can shake up the medical field because of their high specificity, high sensitivity, high speed, cost-effectivity, and no need for highly trained personnel and advanced laboratories. In Urology, these methods can be used for various applications such as liquid biopsy and rapid detection of prostate or bladder cancer-specific mutations in cell-free DNAs. 

Acknowledgments

None.

Conflict of Interest

The author declares that there is no conflict of interest.

Funding

There is no funding.

Ethical statement

Not Applicable.

Data Availability

Data will be provided on request.

Abbreviations

CAS                   CRISPR associated protein

CRISPR             Clustered regular interspaced short palindromic repeats

DETECTR         DNA endonuclease targeted CRISPR trans reporter

RPA                    Recombinase polymerase amplification

SHERLOCK      Specific high sensitivity enzymatic reporter unlocking