Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks

Abstract

Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.

Exploring nano-enabled CRISPR-Cas-powered strategies for efficient diagnostics and treatment of infectious diseases

“…CRISPR/Cas9-triggered nicking endonuclease-mediated strand displacement amplification (CRISDA) was developed in addition to the fluorescence technique for susceptible multiplication and identification of double-stranded DNA (dsDNA), and this method takes complete privilege of the increased selectivity, restrictive precision, and distinct morphological rearrangements of CRISPR signaling pathways in recognizing the target DNA. This employs Cas9 protein to sense a relatively specific target and create unique nicks in quasi-DNA that are subsequently replicated in vitro by DNA polymerases [125,126]. Cas9 induced exponentially amplified response (Cas-EXPAR), a novel site-specific Cas9based nucleotide detection method, was developed in 2018, based on nicking action of target-specific Cas9's nucleases [127].…”

CRISPR Approaches for the Diagnosis of Human Diseases

“…The combination of Cas9 activity with other techniques, such as nucleic acid amplification, is being used for the detection of specific DNA and RNA sequences, thus applying it as a biosensing system for diagnostic purposes. The applications of Cas9-based biosensing systems can be classified into two categories: those leveraging the specific target cleavage activity of the Cas9 effector, and those combining the target-specific binding activity of nuclease-deficient Cas9 (dCas9) with split signal-transducing proteins [45]; such biosensors can be applied to genotyping pathogens and discriminating single nucleotide polymorphisms (SNPs). In 2016, Pardee and collaborators successfully combined Cas9 cleavage activity with nucleic acid amplification technologies, such as nucleic acid sequence-based amplification (NASBA), and an isothermal amplification technique based on CRISPR cleavage (NASBACC).…”

The Viral Threat in Cotton: How New and Emerging Technologies Accelerate Virus Identification and Virus Resistance Breeding

“…After completing the virus sequencing step, it is possible to develop new sensitive virus detection techniques and replace costly qRT-PCR-based diagnosis testing systems ( Srivastava et al, 2020 ). Some accurate and ultrasensitive nucleic acid-based testing (NAT) tools in development for multiplexed virus infection capable of replacing qRT-PCR are recombinase polymerase amplification (RPA; Kim et al, 2018 ), loop-mediated isothermal amplification (LAMP; Selvaraj et al, 2019 ), and clustered regularly interspaced short palindromic repeats (CRISPR; Kellner et al, 2019 ).…”

“…Among CRISPR-NAT tools, CAS9 has shown to be an ultrasensitive pathogen detection method. Converting the developing NAT to point-of-care testing (POCT) will drastically reduce costs and accelerate viral identification due to on-site identification and easy handling ( Srivastava et al, 2020 ). CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA; Wang et al, 2020 ) and Editor-Linked Uniform Detection Assay (FELUDA; Azhar et al, 2021 ) are new POCT tools optimized for fast and easy in situ screening of human viruses using paper strips.…”