CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review

Abstract

Recently, CRISPR-Cas system-based assays for bacterial detection have been developed. The aim of this scoping review is to map existing evidence on the utilization of CRISPR-Cas systems in the development of bacterial detection assays. A literature search was conducted using three databases (PubMed, Scopus, and Cochrane Library) and manual searches through the references of identified full texts based on a PROSPERO-registered protocol (CRD42021289140). Studies on bacterial detection using CRISPR-Cas systems that were published before October 2021 were retrieved. The Critical Appraisal Skills Programme (CASP) qualitative checklist was used to assess the risk of bias for all the included studies. Of the 420 studies identified throughout the search, 46 studies that met the inclusion criteria were included in the final analysis. Bacteria from 17 genera were identified utilising CRISPR-Cas systems. Most of the bacteria came from genera such as Staphylococcus, Escherichia, Salmonella, Listeria, Mycobacterium and Streptococcus. Cas12a (64%) is the most often used Cas enzyme in bacterial detection, followed by Cas13a (13%), and Cas9 (11%). To improve the signal of detection, 83% of the research exploited Cas enzymes’ trans-cleavage capabilities to cut tagged reporter probes non-specifically. Most studies used the extraction procedure, whereas only 17% did not. In terms of amplification methods, isothermal reactions were employed in 66% of the studies, followed by PCR (23%). Fluorescence detection (67%) was discovered to be the most commonly used method, while lateral flow biosensors (13%), electrochemical biosensors (11%), and others (9%) were found to be less commonly used. Most of the studies (39) used specific bacterial nucleic acid sequences as a target, while seven used non-nucleic acid targets, including aptamers and antibodies particular to the bacteria under investigation. The turnaround time of the 46 studies was 30 min to 4 h. The limit of detection (LoD) was evaluated in three types of concentration, which include copies per mL, CFU per mL and molarity. Most of the studies used spiked samples (78%) rather than clinical samples (22%) to determine LoD. This review identified the gap in clinical accuracy evaluation of the CRISPR-Cas system in bacterial detection. More research is needed to assess the diagnostic sensitivity and specificity of amplification-free CRISPR-Cas systems in bacterial detection for nucleic acid-based tests.

Exploiting the Specificity of CRISPR/Cas System for Nucleic Acids Amplification-Free Disease Diagnostics in the Point-of-Care

“…They exhibit a miniaturized structure comprising 400–700 amino acids, about half of the size of Cas9 and Cas12. Cas14 has been re-classified into the Cas12 family due to its similarity with Cas12, which depends on T-rich PAM and utilizes dsDNA or ssDNA as its target. , Cas14 demonstrated excellent detection and discrimination between identical ssDNA sequences with one nucleotide variation comparable to Cas12a, even in the presence of a PAM sequence . We can expect Cas14 to be widely repurposed for amplification-free diagnostic applications to identify ssDNA SNPs without needing a PAM sequence due to their increased specificity and convenience.…”

Applicability of Smart Tools in Vegetable Disease Diagnostics

“…Depending on the price of the final product, the utilization of such tools may be widespread. However, Selvam et al [87] noted that more research on specificity and sensitivity in individual cases must be performed. Some authors have highlighted that most CRISPR/Cas-based diagnostics are not suited to quantitative detection.…”

CRISPR-Cas System: A Tool to Eliminate Drug-Resistant Gram-Negative Bacteria

“…Apart from the Cas9 enzymes, Cas proteins belonging to other types have also been used in the detection system. The Cas12a and its orthologues (LbCas12a, LbaCas12a from Lachnspiraceace bacterium ND2006 and FnCas12a from Francisella novicida U112) have been used most widely for bacterial detection followed by the Cas13a and its orthologues (Lbu-Cas13a from Leptotrichia buccalis, LwaCas13a and LwCas13a from Leptotrichia wadei) [121]. Qui et al, used the CRISPR Cas12a technology to develop the CRISPR-HP assay for the detection of H. pylori from stool specimens.…”