CRISPR System for Covid-19 Detection

The recent Corona Virus, which is known as the Covid-19 virus, first emerged in December 2019 and rapidly spread to all parts of the world, causing a global pandemic. The high number of cases of the virus in all countries of the world and its associated mortality has become a source of great concern and leading to a number of studies being conducted to evaluate what will help control the spread. Experts claim that the early detection of the virus is the key to allow an early treatment plan and prevent the spread of the virus to those around the affected individual. Even though the PCR test is the current standard diagnostic test for the Covid-19 virus all over the world, it has its limitations, and there is now a need for a diagnostic system that is much more efficient in the detection of the virus, in turn helping control the morbidity and mortality associated with it.

What is the CRISPR System?

The CRISPR System allows alteration of gene function by enabling the researchers to make changes in specific genomic sequences in the DNA. This system essentially works as a pair of molecular scissors which allows the cutting of DNA strands to get the desires results.

This system can be divided into two classes, known as class 1 and class 2, which are further composed of different types of CRISPR systems.

Class 1

• Type 1: The Type 1 CRSPR system contains Cas3 nuclease, which allows cutting of DNA to achieve desired results. 
• Type III: The Type III CRISPR system contains Cas10 nuclease, which allows the cutting of specific RNA strands.
• Type IV: Type IV CRISPR system can be divided into two further subtypes, which contain the Cas7, Cas5, and smaller variants of Cas8.

Class 2

• Type II: The Type II CRISPR system consists of Cas9 endonuclease, which allows the cutting of DNA.
• Type V: Type V CRISPR system contains Cas12, which is used to cut DNA.
• Type VI: Type VI CRISPR consists of Cas13 nuclease, which allows the specific cut to be made into the target RNA sites. (1)

Covid-19 Detection with CRISPR/CAS 12

DETECTR, which stands for SARS-Cov-2 DNA Endonucease targeted CRISPR trans reporter, is a type of CRISPR/Cas12 system that was created by Broughton et al. for the diagnosis of the virus. This fairly simple technique allows rapid diagnosis of the virus in an individual due to its ability to efficiently recognize the SARS-Cov-2 RNA extracts. Studies have shown this diagnostic method to detect the virus from a patient’s respiratory tract within a period of only 40 minutes. 

This diagnostic method allows isothermal amplification and reverse transcription by a process known as loop-mediated replication. This facilitates the production of purified RNA taken from either the nasopharyngeal or oropharyngeal swabs. This is followed by the detection of predetermined virus sequences by Cas12a, after which the reporter molecule undergoes cleavage and reveals the existence of the virus in the taken swab. This diagnostic procedure removes the requirement for thermocycling and isothermal signal amplification, which in turn provides numerous advantages, including faster delivery of test results, target specificity of single nucleotides, allowing user-friendly reporting formats, and no requirement of complicated laboratory systems and setups. (2)

Covid-19 Detection with CRISPR/CAS 13

The CRISPR/Cas13 system also allows efficient diagnosis of the Covid-19 virus by evaluating the base pairing between the crRNA molecule and target sequence. This does not only allow it to be very accurate but also be used as programmable diagnostic equipment. Even though the CRISPR/CAS 13 system is highly efficient, it still requires a supply of extracted nucleic acid for it to be able to carry out its diagnostic process. This may cause its use to be limited, as not everyone has access to sophisticated extraction tools. To overcome these complications, this system was upgraded by Myhrvold et al., where they combined the HUDSON system with the SHERLOCK system. This new system allows chemical reduction and heat to be applied to the nucleases in order to degrade them and cause their breakdown. The breakdown of the RNA then results in the lysis of viral particles, which facilitates the diagnostic procedure. (3)

Covid-19 Diagnosis with CRISPR/ CAS 3

Another CRISPR bases diagnostic system which can be used for detection of the COVID-19 virus and provide accurate diagnosis is the CRISPR/Cas3 system which was developed by Yoshimi et al. The system is referred to as CONAN, which stands for Cas3 operated nucleic acid detection N. This system works in association with Isothermal amplification methods and allows an accurate, fast and device less diagnosis of the Covid-19 virus. 

Benefits of the CRISPR System for Covid-19 Detection

There are numerous benefits of the CRISPR System for the diagnosis of the Covid-19 virus over the conventional methods, which include the antibody and PCR test. The CRISPR diagnostic system does not require thermocycling due to the use of isothermal amplification as part of the system. This also allows it to deliver the results much faster than any other diagnostic tests and allow the individual to start their treatment much sooner, which increases the prognosis of their infection. Moreover, this technique does not require the use of any specialized laboratory tools and equipment and uses accessible reporting techniques, which makes it much more convenient for usage. 

However, one of the most significant advantages of this system is its ability to recognize the numerous infectious disease which may be caused by newly emerging variants of a virus, like the Corona Virus which is causing a global pandemic due to its different forms. The system is able to accurately detect any mutant forms of a virus, which allows early measures to be taken against its spread. 

One challenge that is faced by the viral diagnostic system is the changes in the viral load at different stages of the disease and its ability to impact the test results. For example, when the infection is at a particular stage when the viral loads are exceptionally low, the conventional methods may not be able to detect it; however, that is not the case with the CRISPR diagnostic system. This system is able to efficiently diagnose the presence of the virus at any viral load and stage of the infection as it primarily relies on the detection of the genome of the virus in the system.

References:

1. Rahimi H, Salehiabar M, Barsbay M, et al. CRISPR Systems for COVID-19 Diagnosis [published online ahead of print, 2021 Jan 27]. ACS Sens. 2021;acssensors.0c02312. doi:10.1021/acssensors.0c02312
2. Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan CY, Guevara H, Wadford DA, Chen JS, Chiu CY. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020 Jul;38(7):870-874. doi: 10.1038/s41587-020-0513-4. Epub 2020 Apr 16. PMID: 32300245.
3. Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan AL, Paul LM, Parham LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S, Michael SF, Lorenzana I, Yozwiak NL, MacInnis BL, Bosch I, Gehrke L, Zhang F, Sabeti PC. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018 Apr 27;360(6387):444-448. doi: 10.1126/science.aas8836. PMID: 29700266; PMCID: PMC6197056.