The novel coronavirus, also known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current pandemic that has already infected and claimed the lives of millions. Despite research efforts, scientists continue with their struggle to develop a successful therapeutic strategy and vaccine to control the raging pandemic. However, attempts to develop diagnostic tools have met with varying degree of success. Conventional detection technique for SARS-CoV-2 involves the use of the real-time PCR method (RT-PCR). Research has shown that CRISPR technology has immense potential in the development of diagnostic platforms and therapeutic strategies against SARS-CoV-2 infection.
CRISPR/Cas technique involves the sgRNA directed cleavage of target DNA by Cas protein. Several Cas proteins have been identified, each having their unique functionality. While Cas9 is commonly used for gene editing, Cas12 and Cas13 are commonly employed for diagnosis of diseases.
Recently, CRISPR/Cas13-based SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) technique has been developed for rapid detection of SARS-CoV-2 by Zhang and others from the MIT, Cambridge. The SHERLOCK technique relies on the nucleic acid detection capacity of CRISPR/Cas13 and the use of a quenched fluorescent ssRNA probe. This technique targets the S and ORF lab protein genes within the SARS-CoV-2 genome. The Cas13 randomly inserts breaks into the viral ssRNA around the target genes, producing fluorescence signals. Thus, when a sample containing SARS-CoV-2 genome is tested, the Cas13 gets activated and fluorescence signals corresponding to the nuclease activity of Cas13 is detected. The SHERLOCK technique is rapid and takes about an hour to produce the results.
Similarly, Broughton and a team of scientists from San Francisco recently proposed the detection of SARS-CoV-2 using CRISPR/Cas12 technique. The method utilises CRISPR/Cas12 based lateral flow assay to detect the presence of SARS-CoV-2 genome (E gene and N gene) in the swabs collected from the patients. The technique is rapid and is claimed to produce results within 40 minutes that can be visualised on a lateral flow strip that uses human RNase P gene as a control. This technique of SARS-CoV-2 detection is proposed as an alternative to conventional RT-PCR assay.
In addition to detection of SARS-CoV-2, therapeutic application of CRISPR technique is also being evaluated. Timothy Abbott and his team from the Stanford University have developed PAC-MAN (prophylactic antiviral CRISPR in human cells) method based on CRISPR/Cas13d capable of disabling and degrading SARS-CoV-2 ssRNA. The Cas13d nuclease is capable of cleaving viral positive-sense RNA and thus inhibiting viral replication and cell cycle. The study identified 22 crRNAs that are capable of targeting the genomes of all known coronaviruses without any mismatch. This indicates the immense prospect of PAC-MAN approach in developed COVID-19 therapy and vaccination.
Similarly, Nalawansha and Samarasinghe from the Yale University, United States have proposed AntiBody And CAS fusion (ABACAS) approach to treat SARS-CoV-2 infection. The approach uses Cas13along with an antibody fragment specific to the S-protein of SARS-CoV-2. The antibody would recognise the S-protein and help to deliver Cas13 into the infected host cells. Upon selective delivery of the CRISPR component, Cas13 will cleave the viral ssRNA. This method promises to reduce off-tissue effects that might be associated with the PAC-MAN approach.