Advancements in CRISPR-Based Biosensing for Next-Gen Point of Care Diagnostic Application

Abstract

With the move of molecular tests from diagnostic labs to on-site testing becoming more common, there is a sudden rise in demand for nucleic acid-based diagnostic tools that are selective, sensitive, flexible to terrain changes, and cost-effective to assist in point-of-care systems for large-scale screening and to be used in remote locations in cases of outbreaks and pandemics. CRISPR-based biosensors comprise a promising new approach to nucleic acid detection, which uses Cas effector proteins (Cas9, Cas12, and Cas13) as extremely specialized identification components that may be used in conjunction with a variety of readout approaches (such as fluorescence, colorimetry, potentiometry, lateral flow assay, etc.) for onsite analysis. In this review, we cover some technical aspects of integrating the CRISPR Cas system with traditional biosensing readout methods and amplification technologies such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) and continue to elaborate on the prospects of the developed biosensor in the detection of some major viral and bacterial diseases. Within the scope of this article, we also discuss the recent COVID pandemic and the numerous CRISPR biosensors that have undergone development since its advent. Finally, we discuss some challenges and future prospects of CRISPR Cas systems in point-of-care testing.

Keywords: CRISPR; Cas effector proteins; E-CRISPR; biosensor; diseases.

Figure 1

Step mechanism used by the CRISPR-Cas system to identify, isolate, and/or knockout a gene sequence. (A) Target-specific binding: The process of acquiring a spacer starts with Cas1 and Cas2 recognizing the invasive DNA and cleaving a protospacer. In order to replicate the direct repeat and repair the CRISPR, the protospacer is ligated to the direct repeat next to the leader sequence. The Cas protein that is catalytically inactive is bent to the target gene, which is complementary to gRNA. (B) Biogenesis and target-specific cleavage: The CRISPR sequence must be converted into CRISPR-RNA (crRNA), which later directs the Cas nuclease to the target during the interference stage. The crRNA is first produced as a single lengthy transcript that covers the majority of the CRISPR array. The Cas proteins then split this transcript into crRNAs. The way that different CRISPR-Cas systems create crRNAs varies. The target gene is cleaved by Cas proteins thatare followed by sequence-specific binding. (C) Sequence-specific trans cleavage: The Cas protein non-specifically cleaves the ssDNA or ssRNA upon binding to the target gene, thereby freeing the reporter probe.

Figure 2

Three widely used signal detection techniques can be monitored to detect the existence of the target gene. (A) Fluorescence-based sensing uses target sequences prepared with fluorescent probes to detect target sequences based on the changes in fluorescence intensity. A higher intensity than the control denotes a positive result; examples are SHERLOCK and DETECTR. (B) Colorimetric sensing uses color-changing complexes attached to CRISPR-Cas as complexes to detect target sequences. When the complexes are cleaved using targeted the CRISPR assay (Cas9), a color change is observed. (C) Electrochemical sensing detects the target sequence based on changes in electrical potential using a potentiostat.

Figure 3

Five-step workflow of CRISPR-based detection system to detect SARS-CoV-2 virus. (1) Sample collection using nasal or oral swabs. (2) RNA molecules are extracted and isolated from the sample, as SARS-CoV-2 is an RNA-based virus. (3) Isolation is followed by an amplification procedure such as RT-PCT, RT-RPA, or RT-LAMP to increase the LOD, (4) followed by detection using the CRISPR-Cas system and (5) detection using fluorescence, colorimetry, electrochemical, or other sensory readout methods.