Current Knowledge on CRISPR Strategies Against Antimicrobial-Resistant Bacteria

Abstract

CRISPR/Cas systems have emerged as valuable tools to approach the problem of antimicrobial resistance by either sensitizing or lysing resistant bacteria or by aiding in antibiotic development, with successful applications across diverse organisms, including bacteria and fungi. CRISPR/Cas systems can target plasmids or the bacterial chromosome of AMR-bacteria, and it is especially necessary to have an efficient entry into the target cells, which can be achieved through nanoparticles or bacteriophages. Regarding antibiotic development and production, though the use of CRISPR/Cas in this field is still modest, there is an untapped reservoir of bacterial and fungal natural products, with over 95% yet to be characterized. In Streptomyces, a key antibiotic-producing bacterial genus, CRISPR/Cas has been successfully used to activate silent biosynthetic gene clusters, leading to the discovery of new antibiotics. CRISPR/Cas is also applicable to non-model bacteria and different species of fungi, making it a versatile tool for natural products discovery. Moreover, CRISPR/Cas-based studies offer insights into metabolic regulation and biosynthetic pathways in both bacteria and fungi, highlighting its utility in understanding genetic regulation and improving industrial strains. In this work, we review ongoing innovations on ways to treat antimicrobial resistances and on antibiotic discovery using CRISPR/Cas platforms, highlighting the role of bacteria and fungi in these processes.

Keywords: AMR; BGCs; CRISPR/Cas; Streptomyces; antibiotic; multidrug-resistant bacteria; nanoparticles; phages.

Figure 1

Molecular principles of CRISPR genome editing. CRISPR genome editing relies on RNA-guided nucleases such as Cas9 and Cas12a for site-specific target DNA recognition and cleavage. Cas9 utilizes a dual-guide RNA composed of a CRISPR RNA (crRNA)-trans-activating CRISPR RNA (tracrRNA) pair or a single-guide RNA (sgRNA), whereas Cas12a is programmed with a crRNA only. Target DNA recognition is dependent on complementarity with the spacer sequence of the guide RNA as well as the presence of a protospacer adjacent motif (PAM). Cas9 recognizes an NGG PAM, whereas Cas12a requires a TTTV PAM (V = G, C, or A). Upon target binding, the nucleases catalyze DNA cleavage, generating a DNA double-strand break (DSB). DSB repair by cellular DNA repair pathways leads to the introduction of genetic modifications (edits). The end-joining pathways result in short insertions or deletions (indels), whereas homology-directed repair (HDR) using an exogenous DNA repair template can be used to engineer precise modifications. The green and blue colours in the figure show homology with a repair template, and the red and yellow colours represent a random sequence and a template sequence, respectively. Reproduced from [42] under a CC BY 4.0 license.

Figure 2

Overview of the two CRISPR/Cas strategies to combat antibiotic-resistant bacteria: AMR management and antibiotic research and production.