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.