Sensitive and specific nucleic acid detection is crucial for clinical diagnostics, genotyping, and biotechnological advancements. Current methods of nucleic acid detection however, either lack the sensitivity or the specificity to detect nucleic acids at low concentrations and/or are too expensive, time-consuming, and complex to use outside of standard laboratories. Recently scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar level.
The Zhang lab has utilized the natural RNAse activity of the Cas13 protein to develop and optimize the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2) (Gootenberg et al., 2017 and 2018). While the Doudna lab has utilized Cas12a’s non-specific ssDNA degradation to develop the method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018).
Both SHERLOCK and DETECTR harness the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a respectively to cleave and activate a reporter. The detectable signal from this reporter can be measured and quantified to determine the presence and quantity of DNA, RNA or a mutation of interest. Together SHERLOCK and DETECTR demonstrate the power of CRISPR-based diagnostics.
SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING
Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNAse (Abudayyeh et al 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave ssRNA or mRNA, similarly to RNA interference, providing an exciting use of CRISPR for gene knockdown without genome editing. Cas13, as previously stated, also exhibits a “collateral effect” of non-specific ssRNA cleavage. In SHERLOCK the promiscuous RNAse activity of Cas13, with a quenchable ssRNA reporter, is combined with an isothermal amplification step to rapidly detect a single molecule of DNA or RNA.
How does SHERLOCK work?
Cas13 can be programmed with crRNA to target an ssRNA of interest. Recognition and binding of Cas13 to the programmed sequence activates Cas13’s promiscuous cleavage of surrounding ssRNA molecules. In SHERLOCK a quenched fluorescent ssRNA reporter is added to the reaction. Cleavage of the quenchable fluorescent RNA by the “activated” Cas13 produces a quantifiable signal that indicates the presence of your targeted nucleic acid. To increase the sensitivity of the assay, targeted DNA or RNA from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-RPA, respectively. RPA is coupled with T7 transcription to convert amplified DNA to RNA for subsequent detection by Cas13. This amplification step in combination with the ssRNA reporter enables SHERLOCK to detect DNA or RNA with atomolar sensitivity and single base pair mismatch specificity. The Zhang lab demonstrated that SHERLOCK could reliably distinguish between Zika and a closely-related virus, Dengue from multiple sample sources. SHERLOCK could also detect low-frequency cancer mutations from cell-free DNA fragments as well as health-related single nucleotide polymorphisms (SNPs) from human saliva.
SHERLOCK had a few limitations when it was introduced in 2017. The first being quantitation, as the system relies on the exponential pre-amplification of DNA, which can saturate reporters. SHERLOCKv2 uses far less primer in the pre-amplification step allowing for greater quantitation without compromising sensitivity. Scientists also used the cleavage preferences of multiple Cas enzymes to create a multiplex platform. In this platform, Cas enzymes, such as variations of Cas13 and Cas12a, are combined in one reaction with different fluorescent reporters to accurately detect multiple targets. In addition, Csm6, a CRISPR type-III effector nuclease, can be used in conjunction with Cas13 to amplify the signal of a single target. Csm6 can cleave ssRNA complementary to a crRNA of interest (Niewoehner and Jinek, 2016) and is conveniently activated by Cas13 ssRNA cleavage byproducts. Thus, binding of Cas13 to the programmed region of interest would lead to both the cleavage of a Cas13 specific reporter and the activation of Csm6. The Zhang lab showed that, by adding Csm6 and a Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter) to the assay, they could significantly amplify the detection signal for a single target.
The second limitation of SHERLOCK was its reliance on fluorescence, which requires additional equipment to acquire data. SHERLOCKv2 was adapted so that a cleaved reporter could be detected on commercial lateral flow strips, similarly to pregnancy test. With lateral flow strips, the presence of your DNA or RNA of interest within a given sample is simply determined by the number of bands present on the strip. This type of readout allows for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly, providing reliable results in as little as an hour.
Applications of SHERLOCK
SHERLOCKv2 provides a method to detect nucleic acids with high sensitivity and specificity without compromising speed, ease of use, and portability. This method can be used for an array of applications including clinical diagnostics (e.g. pathogen or virus detection), therapeutics and sensitive genotyping. The beauty of SHERLOCKv2 is that it can be used easily and effectively in the lab and in the field.
DETECTR- DNA Endonuclease Targeted CRISPR Trans Reporter
Cas12a (Cpf1) is a CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche et al., 2015 ). Cas12a however recognizes a different PAM site, catalyzes its own guide RNA maturation, and generates 5’ and 3’ staggered ends after dsDNA breaks; making it attractive for gene editing. The Doudna lab, while investigating the Cas12a enzyme, discovered Cas12a’s ability to cleave non-specific (trans) ssDNA after being targeted to a DNA of interest via a crRNA. Cas12a’s non-specific ssDNA trans-cleavage exhibits multiple turnover events (~1250 per second) when bound to a crRNA-complementary dsDNA activator. The Doudna lab used this rapid and indiscriminate DNase activity of Cas12a to develop a highly sensitive and selective DNA detection platform, DETECTR.
How does DETECTR work?
DETECR works similarly to SHERLOCK. Cas12a is targeted to a specific DNA sequence, such as the Human papilloma virus (HPV) genome, via a crRNA. An ssDNA- fluorescently quenched (FQ) reporter, which will produce a signal when the ssDNA is degraded, is added to the reaction. To enhance sensitivity DNA is first amplified through isothermal amplification by RPA. When Cas12a-cRNA base pairs with the dsDNA of interest, the DNase activity of Cas12a is initiated. Surrounding trans-ssDNA, including the ssDNA-FQ reporter are subsequently degraded. A quantifiable fluorescent signal designates the presence of your DNA of interest, in this case HPV. As proof of concept, Chen et al., 2018 demonstrated that DETECTR could accurately distinguish between two similar types of HPV, 16 and 18, from human cells, at atomolar levels, within one hour. DETECTR thus has the ability to rapidly detect nucleic acids with high selectivity and sensitivity from patient samples.
Applications of DETECTR
DETECTR allows for simple and efficient detection of nucleic acids in a mixed population for an array of molecular and clinical diagnostic applications. The company Mammoth Biosciences has been started based on DETECTR technology with the mission to “provide a CRISPR-based platform on which an infinite number of tests” for biosensing can be built.
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2. Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 6245(February), 1–8. PubMed PMID: 29449511.
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4. Gootenberg, J. S., Abudayyeh, O. O., Kellner, M. J., Joung, J., Collins, J., & Zhang, F. (2018). Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 0179 (February), 1–10. PubMed PMID: 29449508. PubMed Central PMCID: PMC5961727.
5. Niewoehner, O., & Jinek, M. (2016). Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. Rna, 22(3), 318–329. PubMed PMID: 26763118. PubMed Central PMCID: PMC4748810.
6. Shmakov, S., Abudayyeh, O. O., Makarova, K. S., Wolf, Y. I., Gootenberg, J. S., Semenova, E., … Koonin, E. V. (2015). Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell, 60(3), 385–397. PubMed PMID: 26593719. PubMed Central PMCID: PMC4660269.
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Additional resources on the Addgene Blog
- Learn about using toehold switches to detect Zika virus
- Learn more about targeting RNA with Cas13a (C2c2)
- Learn more about Cas12a (Cpf1) and its multiplex genome editing abilities
Resources on Addgene.org