Mitigating Antibiotic Resistance: The Utilization of CRISPR Technology in Detection

Abstract

Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation.

Keywords: CRISPR; antibiotic detection; non-nucleic acid targets; nucleic acid targets.

Figure 2

The CRISPR/Cas system-based detection platform for antibiotic-resistance genes. (A) Specific DNA was cut using the Cas9 enzyme, the fluorescent dye was added, and then the plasmid was stretched in the nano-channel, and the information was observed by fluorescence microscope. Copyright Vilhelm Müller. (B) The resistance gene fragment was amplified by RPA technology to activate the CRISPR/Cas12a system, followed by colorimetric detection using Au-Fe₃O₄ nanoenzymes.(* p < 0.05, ** p < 0.01) Reproduced with permission from Ref. [108]. Copyright 2023 Elsevier. (C) CRISPR/Cas9 recognizes and cleaves drug-resistant genes and activates IEXPAR amplification for rapid detection. Reproduced with permission from Ref. [106]. Copyright 2022 Elsevier. (D) The presence of target ARGs activates CRISPR/Cas12a to degrade the crosslinker, making the solution red. Without target genes, it turns purple due to the undegraded crosslinker. Reproduced with permission from Ref. [110]. Copyright 2023 American Chemical Society.

Figure 3

CRISPR/Cas-based detection platform for antibiotics. (A) The AMP aptamer releases an activator upon target binding, activating Cas14 to cleave a probe, and AMP concentration is quantified by ICPMS detection of terbium isotope intensity. Used with permission of © The Royal Society of Chemistry 2021, from [128]. Permission conveyed through Copyright Clearance Center. (B) Tobramycin-bound aptamer triggers SDA to generate ssDNA activators that activate CRISPR/Cas12a to cleave reporter probes and output signals. Reproduced with permission from Ref. [42]. Copyright 2021 Elsevier. (C) In the presence of the ligand, dissociation of the aTF allows transcription of the CRISPR array, activating CRISPR/Cas12a and cleaving the probe, outputting a signal. Copyright©2022 Ahmed Mahas. Published by American Chemical Society. This publication is licensed under CC-BY 4.0. (D) The aptamer recognizes ampicillin and releases the ssDNA activator, which activates the trans-cleavage of Cas12a and outputs a fluorescent signal. Reproduced with permission from Ref. [41]. Copyright 2023 Elsevier. (E) Without kanamycin, S1 triggers HCR1, generating a strong electrical signal. In the presence of kanamycin, activation of CRISPR/Cas12a blocks HCR1 and reduces the electrical signal. Reproduced with permission from Ref. [132]. Copyright 2024 Elsevier.

Figure 1

Non-nucleic acid target-detection strategy-based on CRISPR-Dx. The innermost circle describes the principle of action of the four CRISPR effector proteins; the middle circle represents the non-nucleic acid targets detectable by the CRISPR effector proteins; and the outermost circle describes the non-nucleic acid targets in the environment that are converted by the bio-transduction element into recognizable nucleic acid signals, which are then outputted on the constructed CRISPR detection platform. Created in https://BioRender.com.