Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

Emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. We established a series of CRISPR-Cas13a-based antimicrobials, termed PhagoCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus through promiscuous RNA cleavage after recognizing corresponding antimicrobial resistance genes. PhagoCas13a constructs were generated by packaging CRISPR-Cas13a into a bacteriophage to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the PhagoCas13a(s) exhibited strong bacterial killing activities upon recognizing target genes regardless of their location. The antimicrobials’ treatment efficacy was confirmed using a Galleria mellonella larvae model. Further, we demonstrated that the PhagoCas13a(s) can assist in bacterial gene detection without employing nucleic acid amplification and optical devices. One Sentence Summary Novel gene-specific antimicrobials capable of killing drug-resistant bacteria and applicable to detect bacterial genes were developed.