The Application of the CRISPR-Cas System in Antibiotic Resistance

Abstract

The emergence and global epidemic of antimicrobial resistance (AMR) poses a serious threat to global public health in recent years. AMR genes are shared between bacterial pathogens mainly via horizontal gene transfer (HGT) on mobile genetic elements (MGEs), thereby accelerating the spread of antimicrobial resistance (AMR) and increasing the burden of drug resistance. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are an RNA-guided adaptive immune system in prokaryotes that recognizes and defends against invasive genetic elements such as phages and plasmids. Because of its specifically target and cleave DNA sequences encoding antibiotic resistance genes, CRISPR/Cas system has been developed into a new gene-editing tool for the prevention and control of bacterial drug resistance. CRISPR-Cas plays a potentially important role in controlling horizontal gene transfer and limiting the spread of antibiotic resistance. In this review, we will introduce the structure and working mechanism of CRISPR-Cas systems, followed by delivery strategies, and then focus on the relationship between antimicrobial resistance and CRISPR-Cas. Moreover, the challenges and prospects of this research field are discussed, thereby providing a reference for the prevention and control of the spread of antibiotic resistance.

Keywords: CRISPR-Cas; antibiotic resistance; horizontal gene transfer.

Figure 1

The structure of CRISPR system.

Figure 2

The working mechanism of the CRISPR-Cas system. The bacterial defense mechanism of the CRISPR/Cas systems includes three stages: Adaptation stage: acquisition of spacer sequences; Expression stage: Generation of the crRNA and Cas protein; Interference stage: crRNA-guided nucleic acid-targeted cleavage.

Figure 3

(A) The methods for delivery of CRISPR-Cas components: plasmids-based delivery (Design and synthesize sgRNA targeting the target gene and ligate it into a plasmid vector containing Cas9); phage-based delivery (The CRISPR-Cas system was integrated into the phage genome and delivered with the phage as a vector); Extracellular vesicles(EVs)-based delivery and nanoparticle-based delivery (Cas9 protein and sgRNA form a ribonucleoprotein (RNP) complex and packaged into EVs and nanoparticles). (B) The application of CRISPR-Cas system in antibiotic resistance. When the delivery of CRISPR-Cas systems in the bacteria cells, the antibiotic-resistant genes (ARG) on the plasmids could be eliminated and the bacteria can re-sensitized against antibacterial agents. CRISPR-Cas also shows a strong bactericidal activity and make the cell die after the recognition of the target ARG on plasmid and the target site on genome.