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

Kotaro Kiga,Bintao Cui,Longzhu Cui,Masato Suzuki,Moriyuki Kawauchi,Tanit Boonsiri,Yoshifumi Aiba,Teppei Sasahara,Feng-Yu Li,Aa Haeruman Azam,Yusuke Sato’O,Kanate Thitiananpakorn,Yusuke Taki,José R Penadés,Shinya Watanabe,Rodrigo Ibarra-Chávez,Xin-Ee Tan

doi:10.1038/s41467-020-16731-6

Abstract

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid 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 CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

Highlights

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials
We here report success in developing sequence-specific antimicrobials by packaging the LshCas13a into bacteriophage capsids, which can be used as both therapeutic agents against AMR bacterial infections and nonchemical agents to detect bacterial genes for diagnosis
The CRISPR-Cas[9] decreased the number of bacteria by three orders of magnitude against the bacteria carrying the blaIMP-1 only on the chromosome, but not on the plasmid, when compared with their respective nontargeting controls (Fig. 1b, c). These results agreed with the fact that while CRISPR-Cas[9] caused cell death by double-strand DNA breaks, CRISPR-Cas13a induced cell dormancy by collateral nonspecific cleavage of RNA

Summary

Introduction

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. There is an urgent need for new strategies to develop alternative therapeutic approaches to prevent infections of AMR bacteria. To this end, various nucleic acid-based antibacterials, peptides, bacteriophage therapies, antibodies, bacteriocins, and anti-virulence compounds have been recently developed[5]. We here report success in developing sequence-specific antimicrobials by packaging the LshCas13a into bacteriophage capsids, which can be used as both therapeutic agents against AMR bacterial infections and nonchemical agents to detect bacterial genes for diagnosis

Methods

Results

Conclusion