Next-Gen Diagnostics: Comparing SHERLOCK, DETECTR, and Other CRISPR-Based Platforms

Molecular diagnostics are a very highly changing landscape with the advent of CRISPR-based solutions, and it is fast, accurate, field-applicable. The notable inventions that have appeared among them are SHERLOCK and DETECTR as two of the most promising CRISPR diagnostic systems. Both mechanisms take advantage of the specificity of CRISPR-associated proteins and the ease of isothermal amplification, and the mechanism and sensitivity are different, the targeted disease is different, and the complexity of the operations is different.

This paper will benefit on the comparison of SHERLOCK by the Broad Institute of MIT and Harvard and DETECTR, the first CRISPR-based diagnostic platform led by Mammoth Biosciences, among other CRISPR-based diagnostic platforms. We shall look at how they conduct their work, how they perform and what they may do in a clinical and field aspect.

Coming into Being of CRISPR-Based Diagnostics

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology which was initially known due to the potential of its gene-editing capabilities has now been put into an effect in diagnostics and has found a revolutionary application. CRISPR diagnostics depend on the capacity of CRISPR-related (Cas) proteins to recognize and bind definite nucleic acid chains.

Of crucial importance to the successful performance of those diagnostics is their readiness in combination with isothermal amplification variants, which can quickly multiply genetic material using just a single temperature, like Recombinase Polymerase Amplification (RPA) or Loop-mediated Isothermal Amplification (LAMP). Such methods do not require high-tech thermal cycling machinery, like that needed to perform traditional PCR.

A Summary of the Top CRISPR Diagnostic Technology The following is an overview of the top CRISPR diagnostic systems.

SHERLOCK: Particular High Sensitivity Enzymatic Reporter Unlocking

SHERLOCK is a CRISPR diagnostic platform that relies on the Cas13 enzymer that has an RNA target. When a Cas13 protein and target RNA hybridize, the Cas13 protein shows collateral cleavage activity i.e, it non-specifically cleaves a nearby single-stranded RNA reporter, leaving a measurable signal, usually fluorescence.

Key Features:

Detection Mechanism The detection mechanism is the RNA targeting with collateral cleavage by Cas13.

• Amplification Process: recombinase polymerase amplification (RPA) or alternate amplification process that is isothermal.
• Readout: Fluorescent or lateral flow strip, thus allowing both a laboratory and a point of care.
• Sensitivity: Attomolar sensitivity; has the ability to detect single copy of viral RNA.
• Uses: SHERLOCK has been applied in the identification of viruses like the Zika, the Dengue and notably, SARS-CoV-2.

Strengths:

• Ultra high sensitivity.
• Multiplexing (multiple targets detection).
• Flexibility of portable diagnosing devices.

DETECTR: is a CRISPR trans reporter, DNA endonuclease targeted

DETECTR, invented by Mammoth Biosciences uses Cas12 enzyme that binds specifically to DNA sequences. Cas12 also shows collateral cleavage on recognition of its DNA target, but acts on the close single-stranded DNA reporters.

Key Features:

• Detection Mechanism- Cas12 DNA targeting and collateral cleavage.
• Method of amplication; Isothermic amplification through LAMP.
• Readout: picture outcomes by means of lateral flow strips or fluorescing reporters.
• Sensitivity: femtomolar to attomolar sensitivities.
• Applications: After fast COVID-19 testing, DETECTR received a lot of attention but has a wider applicability in the detection of HPV and other pathogens.

Strengths:

• Quick testing (approximately 30-40 min of sample to result).
• Simple visual displays to deploy into field deployable environments.
• Reverse transcriptase in DNA -compatible (and RNA, by extension).

Comparison of Side-by Side: SHERLOCK vs. DETECTR

Isothermal integration with Amplification

The sensitivity and the speed can be maximized with both SHERLOCK and DETECTR as they incorporate isothermal amplification. Compared to PCR, faster isothermal methods such as RPA and LAMP that amplify at a single, fixed temperature allow such CRISPR diagnostics to be adapted especially for low-resource settings.

So, why does it isothermal amplification? The reason is that it is easy to incorporate isothermal amplification into a GP.

• Portability: there is no requirement of a complex thermal cyclers.
• Speed: It provides results less than an hour.
• Energy Efficiency: It is perfect to use in field settings where there are not guaranteed access to electricity.

Such ease of application is the reason why SHERLOCK and DETECTR are further used in point-of-care (POC) diagnostics and outbreak response, particularly, where there is weak laboratory infrastructure.

Other worth to watch CRISPR-Based Diagnostic Platforms

HOLMES (One-hour Low cost Multipurpose Highly efficient System)

Developed By: Lab of Feng Zhang (Broad institute)

• Su Anzuelo: Enzyme: Cas12a
• Target: DNA viruses, mutations in the genes.
• Characteristics: Has a feat of fast detection in an hour with a similar mechanism of collateral cleavage.

CARMEN (Combinatorial Arrayed Reactions Multiplexed spatiotemporal Evaluation of Nucleic acids)

• Developed By: Broad institute and its partners at MIT.
• Specialty: High throughput multiplex; it is able to measure hundreds of pathogens in one assay.
• Any viral pathogen: Combines microfludics and CRISPR diagnostics to scale pathogen, including many viral pathogens, simultaneously.

Sometimes you might see BRISPR-CRISPR (Bio-Artificial Intelligence of Dual CRISPR-Cas12a)

• Advancement: There is combination of isothermal amplification and CRSIPR detection within a single-tube reaction that reduce the risks of cross contamination.
• Pros: Too easy workflow, very good in POC.

Clinical and Field Applications: On Which Platforms Are the Best?

SHERLOCK’s Sweet Spot:

The high sensitivity and multiplexing that SHERLOCK have ensure that it is the most suitable test in the following areas:

Pandemic Surveillance: Picking out the viruses early.

Complex Diagnostics: detection of multi-pathogen in one sample.

Laboratory Situations: This situation will need the use of fluorescence detection equipment to work most efficiently.

The Advantage offered by DETECTR in the Real-World:

Many DECTECR features include:

•           Point-of-Care: It can easily be used in the clinic, airports, and in households as the lateral flow readouts are visual.

• High Speed of Response: This is particularly useful in responding to diseases that have a high turnaround time e.g. during the COVID-19 pandemic; or HPV test.

•           Field Settings: The minimum of equipment is required, battery-operated heat sources are enough.

Broader Potential:

Other platforms such as HOLMES and AIOD-CRISPR present methods of a similar overall trade-off of speed, ease, and economy, and their potential future in diagnostics of global health challenges.

The existing difficulties in CRISPR-based diagnostics Currently, there are two main issues associated with CRISPR-based diagnostics. The first one is linked to sensor intensity.

Although they have the potential, the CRISPR diagnostic platforms suffer multiple setbacks:

•           Regulatory Approval: Emergency authorizations in the COVID-19 emergency assisted, and broad clinical adoption needs considerably strict authorization.

• Sample Preparation: Minimization of complicated sample handling is an important step towards easiness.

•           Scalability: Although microfluidic advancements through such innovations as CARMEN have demonstrated the resolution of high-throughput requirements, they will not become widely applicable unless they become less expensive and less labor intensive to implement.

Future Look ahead

The development of molecular diagnostic using CRISPR will transform molecular testing by providing:

•           At-Home Testing: Just as it has become easy to test a pregnancy at home, easy-to-use CRISPR kits will soon afford individuals the opportunity of testing a broad category of pathogens.

•           International Availability: CRISPR diagnostics do not rely on heavy infrastructure, so they can connect countries that lack access to testing with middle- to low-income countries.

• Real-Time Outbreak Management: This capability of rapid deployment during an outbreak of the disease could do wonders on the containment process.

With such companies as Mammoth Biosciences developing CRISPR diagnostic technologies further, the world of democratized, affordable testing is only gathering pace.

Conclusion

CRISPR-based molecular diagnostics platforms such as SHERLOCK and DETECTR are taking the lead in bringing the next generation of molecular diagnostics that will transform the global diagnostic market. The strengths of each system also differ: SHERLOCK has unsurpassed sensitivity and the ability to multiplex,” that is, to conduct a wide range of very specific diagnostic tests,” unlike DETECTR, which is user-friendly and can be used quickly in a point-of-care or field setting.

Incorporating into one solution specificity of CRISPR; and the ease of use of isothermal amplification is transforming the way we detect pathogens, genetic mutations, and in the future, even track future pandemics. The emergence of other technologies, including HOLMES, CARMEN, and AIOD-CRISPR, is another indication of abundant opportunities offered by this diagnostic revolution.

In the future, it is possible that the customization of the CRISPR diagnostics in daily clinical activation and mobile diagnostics as well as even at the home levels will emerge as a normal routine- delivery fast, cheap, and precise diagnostics to all the individuals.