CRISPR Technology Applied to the Analysis of Antibiotic Resistance Genes in the Environment.

Environmental transmission of antibiotic resistance poses a risk to human health. Understanding the behavior and health risks of antibiotic resistance genes (ARGs) in the environment requires accurate nucleic acid analyses. Technical challenges remain in the sequencing of ARGs in low abundance relative to total environmental DNA and in on-site analysis. CRISPR technology has been applied to the characterization and monitoring of ARGs in various environmental media. For sequencing of ARGs, CRISPR technology has enabled the enrichment of ARGs present in the environment at a very low abundance. Using CRISPR/Cas9 systems with a pool of guide RNAs (gRNAs) could simultaneously enrich a large panel of ARGs in one environmental sample before sequencing for good coverage and reading depth. CRISPR/Cas9 also enabled targeted long-read sequencing for understanding the genetic context of low-abundance ARGs. CRISPR-based rapid detection has the potential for on-site analysis. Current applications include the analysis of antibiotic-resistant bacteria (ARB) and ARGs in the air and the urban water cycle. CRISPR technology is also useful for differentiating single-nucleotide mutations between antibiotic-susceptible and -resistant variants. Rapid detection of antibiotics together with ARGs is also possible with the use of aptamers that specifically bind to antibiotics.