Programmable Biosensors Based on RNA-Guided CRISPR/Cas Endonuclease

Abstract

Highly infectious illnesses caused by pathogens constitute severe threats to public health and lead to global economic loss. The use of robust and programmable clustered regularly interspaced short palindromic repeat and CRISPR-associated protein (CRISPR-Cas) systems, repurposed from genome-engineering applications has markedly improved traditional nucleic acid detection for precise identification, independently enabling rapid diagnostics of multiplex biomarker with genetic and mutation related to tumors, and microbial pathogens. In this review, we delineate the utility of the current CRISPR-Cas enzyme as biosensors by which these effector toolkits achieve recognition, signaling amplification, and finally, accurate detection. Additionally, we discuss the details of the dominance and hurdles related to expanding this revolutionary technology into an effective and convenient contraption crucial for improving the rational redesign to CRISPR/Cas biosensing. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems by CRISPR/Cas tools.

Emerging Extraction and Diagnostic Tools for Detection of Plant Pathogens: Recent Trends, Challenges, and Future Scope

“…The advent of RNA-guided RNA-targeting CRISPR nuclease Cas13a and subsequent discovery of the other effector proteins Cas12a, Cas13b, and Cas14 have opened newer avenues to promote nucleic acids-based detection techniques . Cas13 effector proteins are suited for direct detection of homologous RNA targets when directed by a suitable RNA sequence, while Cas12 and Cas14 nucleases are fitted for detecting single-stranded and dsDNA targets, respectively . There exist multiple strategies for determining disease resistance in plants through the CRISPR-Cas system: (i) by introducing desired mutations within the coding regions via HDR, (ii) by alterations, deletions, modifications, or inclusion of the cis element in the promoter region, (iii) by knocking-out susceptible factor-encoding genes, (iv) by knocking-out negative regulators involved in defense responses in plants (e.g., TcNPR3), (v) by altering the amino acid sequence of the receptor proteins pertaining to surfaces for evading pathogen effectors (e.g., AtBAK1), and/or (vi) by refinement in key regulators associated with the defense response in plants (e.g., BnWRKY70) .…”

“…Besides their application in plants, CRISPR systems have also been used to target proteins that are known to play key roles in the interaction between fungal and oomycete pathogens and host plants. 47 50 There exist multiple strategies for determining disease resistance in plants through the CRISPR-Cas system: (i) by introducing desired mutations within the coding regions via HDR, (ii) by alterations, deletions, modifications, or inclusion of the cis element in the promoter region, (iii) by knocking-out susceptible factor-encoding genes, (iv) by knocking-out negative regulators involved in defense responses in plants (e.g., TcNPR3), (v) by altering the amino acid sequence of the receptor proteins pertaining to surfaces for evading pathogen effectors (e.g., AtBAK1), and/or (vi) by refinement in key regulators associated with the defense response in plants (e.g., BnWRKY70). 51 Common approaches to execute CRISPR-Cas mediated plant genome editing are via the following ways: (i) transformation of sgRNA along with the Cas nuclease into the plant genome of interest, usually mediated by Agrobacteriummediated transformation (AMT), (ii) DNA-free editing using polyethylene glycol (PEG)-mediated transformation of protoplast by delivering a cargo of ribonucleoprotein (RNP) complex which essentially carries the Cas protein and desired sgRNA into the plant host cell, (iii) RNP complex delivered directly into plant embryo cells through means of biolistic delivery.…”

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

“…There are two classes of CRISPR/Cas systems based on the composition of their effector subunits, Classes 1 and 2 (Table 1 ). Class 1 system contains numerous RNA-effector complexes, and Class 2 system comprises a solitary protein like Cas 9 that conducts all effector complex activities[ 4 ]. The Class 1 CRISPR–Cas system includes type I, III, and putative IV subtypes.…”

“…Over the past few years, several CRISPR/Cas systems belonging to Cas proteins with different characteristics have been developed, which in turn have produced many CRISPR/Cas system-related toolboxes, offering functional robustness, efficiency, and ease of implementation in multiple organisms[ 3 ]. Various gene-editing tools based on CRISPR/Cas9 were introduced in 2013[ 4 ], followed by successful implementation in the modern medical field. Within a few years, CRISPR/Cas technology gradually made crucial breakthroughs and is now widely employed in gene-editing, treatment of genetic diseases, clinical diagnosis of common pathogenic molecules, and alleviating antimicrobial resistance.…”

CRISPR-Based Programmable Nucleic Acid-Binding Protein Technology Can Specifically Detect Fatal Tropical Disease-Causing Pathogens

“…Based on the evolutionary relationships, CRISPR-Cas systems can be grouped into two classes (class I comprises multiple effector proteins while class II has a single crRNA-binding protein), six types, and over 30 sub-types [ 11 ]. Several in-depth reviews have covered the characteristics of different CRISPR-Cas systems [ 6 , 12 , 13 ]. In the case of type II CRISPR-Cas9 system (Cas 9 from Streptococcus pyogenes , S. thermophilus , Staphylococcus aureus , Neisseria meningitidis , and Campylobacter jejuni ), an RNA duplex formed by a trans-activating crRNA (tracrRNA) bound crRNA fuses with the hairpin-rich region of a 20-nucleotide long sgRNA that is complementary to a protospacer region of the target double-stranded DNA (dsDNA) sequence [ 7 ].…”