CRISPR-Based COVID-19 Testing: Toward Next-Generation Point-of-Care Diagnostics

Abstract

As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is an immediate need for a simple, rapid, early and sensitive point-of-care testing for COVID-19 disease. However, current testing approaches do not meet such need. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.

Current molecular diagnostics assays for SARS-CoV-2 and emerging variants

“…It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defence system ( Palaz, Kalkan, Tozluyurt, & Ozsoz, 2021 ). Cas is an enzyme that can recognize and cleave specific target strands of DNA using a guide CRISPR sequence in prokaryotic organisms like bacteria and archaea ( Ganbaatar & Liu, 2021 ). The CRISPR sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote ( Ganbaatar & Liu, 2021 ).…”

“…Cas is an enzyme that can recognize and cleave specific target strands of DNA using a guide CRISPR sequence in prokaryotic organisms like bacteria and archaea ( Ganbaatar & Liu, 2021 ). The CRISPR sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote ( Ganbaatar & Liu, 2021 ). The sequences allow the prokaryote to detect and destroy DNA from similar bacteriophages during subsequent infections, protecting the bacterial cell from invasion.…”

A comprehensive review on current COVID-19 detection methods: From lab care to point of care diagnosis

“…CRISPR is mainly a bacterial defense system against the infected virus. CRISPR RNA (crRNA) guides the CRISPR protein to destroy the target sequence that is attached with a complementary crRNA sequence [ 53 ]. Each CRISPR protein has its signature cleavage ability that makes one unique from the other.…”

“…For instance, Cas9 binds with target dsDNA while Cas13a cleaves SSDNA. Cas14 can recognize ssDNA more efficiently than dsDNA [ 53 ]. The current, global pandemic escalates the interest to use CRISPR tool inaccurate point of care COVID-19 diagnosis.…”

Overcoming the limitations of COVID-19 diagnostics with nanostructures, nucleic acid engineering, and additive manufacturing

“…The purification step was bypassed for some PCR [26] and LAMP [27] protocols, and the 1-copy/μL sensitivity was achieved with LAMP-based assays for detection of SARS-CoV-2 in VTM and saliva [28] . Modified assay protocols for PCR, LAMP, and ELISA were introduced, while alternative assays that utilized enzymatic nucleic acid amplification processes were demonstrated, such as those based upon CRISPR/Cas technology as described in a recent review [29] .…”