CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics

Abstract

COVID-19, the human coronavirus disease caused by SARS-CoV-2, was reported for the first time in Wuhan, China in late 2019. COVID-19 has no preventive vaccine or proven standard pharmacological treatment, and consequently, the outbreak swiftly became a pandemic affecting more than 215 countries around the world. For the diagnosis of COVID-19, the only reliable diagnostics is a qPCR assay. Among other diagnostic tools, the CRISPR-Cas system is being investigated for rapid and specific diagnosis of COVID-19. The CRISPR-Cas-based methods diagnose the SARS-CoV-2 infections within an hour. Apart from its diagnostic ability, CRISPR-Cas system is also being assessed for antiviral therapy development; however, till date, no CRISPR-based therapy has been approved for human use. The Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN), which is Cas 13 based strategy, has been developed against coronavirus. Although this strategy has the potential to be developed as a therapeutic modality, it may face significant challenges for approval in human clinical trials. This review is focused on describing potential use and challenges of CRISPR-Cas based approaches for the development of rapid and accurate diagnostic technique and/or a possible therapeutic alternative for combating COVID-19. The assessment of potential risks associated with use of CRISPR will be important for future clinical advancements.

Keywords: CRISPR; SARS-CoV-2; coronavirus; diagnosis; pandemic (COVID-19); therapeutic.

Figure 1

An overview of the activity of CRISPR-Cas system. The activity of CRISPR-Cas system involves three stages: adaptation, expression-processing, and interference. Adaptation: Nucleic acids of invading pathogens are fragmented into short fragments and inserted between two repeats of CRISPR array as a new spacer by the Cas proteins. Expression/processing: Transcription of CRISPR array in the CRISPR locus occurs to produce pre-crRNA which is processed into mature crRNAs by the Cas nucleases. The Cas9 nuclease binds and stabilizes the *tracrRNA: crRNA duplex and processes pre-crRNA by recruiting RNAse III. Cas12a and Cas13 nucleases process the pre-crRNA by themselves. Interference: Mature crRNAs guide the cleavage of target nucleic acids by CRISPR-Cas effector complexes. The cleavage is based on complementarity between the target sequence and the crRNA. *Trans-activating CRISPR RNA that base pairs with the crRNA to form a functional guide RNA (gRNA).

Figure 2

Schematic representation of dsDNA and ssRNA targeting by Cas12a and Cas13a respectively. CRISPR-Cas12a and CRISPR-Cas13a systems possess dual function viz. processing of pre-CRISPR RNA (pre-crRNA) into mature crRNA and cleavage of target nucleic acid. (A) Cas12a requires T-rich Protospacer Adjacent Motif (PAM) sequence at 5’ end of the Protospacer which is complementary to one strand of the target dsDNA. Cas12a gives a staggered cut on the dsDNA generating 5’ 4-5 nucleotide overhang distal to the PAM site. (B) Cas13a protein recognizes the stem-loop region of crRNA to form Cas13a:crRNA complex and specific cleavage of target ssRNA occurs on the basis of complementarity between the protospacer region and the ssRNA. Cleavage of ssRNA by Cas13a also depends on the presence of 3’ H (A/C/U: Protospacer Flanking Site (PFS)) immediately after the protospacer sequence.

Figure 3

Schematic representation of principles and steps of SHERLOCK (A) and DETECTR (B). (A) SHERLOCK involves amplification of the pathogenic RNA through reverse transcriptase-recombinase polymerase amplification (RT-RPA) followed by in vitro transcription to generate corresponding ssRNA. The ssRNA recognition by Cas13-crRNA complex activates the Cas13 nuclease which further exhibits indiscriminate cleavage of fluorescence tagged reporter ssRNA which is further analyzed on paper strips by lateral flow assay (B) DETECTR amplifies the pathogenic RNA through reverse transcriptase- recombinase polymerase amplification (RT-RPA) or reverse transcriptase- loop-mediated isothermal amplification (RT-LAMP). Target (dsDNA) recognition by Cas12a-crRNA complex activates the Cas12a nuclease which further exhibits indiscriminate cleavage of fluorescence tagged reporter ssDNA which is further analyzed on paper strips by lateral flow assay.

Figure 4

Limitations of CRISPR technology with respect to its therapeutic and diagnostic usage.