Lipid Nanoparticle‐Mediated CRISPR‐Cas13a Delivery for the Control of Bacterial Infection

New formulations of lipid nanoparticles (LNPs) that can deliver nucleic acids to Gram-negative bacteria are proposed to combat bacterial infection. The delivery of nucleic acids by LNPs is aided by LNP-helpers which weaken the bacterial outer membrane. LNPs encapsulating the Cas13a/gRNA expression vector achieve an antibacterial effect in both in vivo and in vitro experiments, suggesting potential for LNPs as a novel platform for the control of bacterial infection.

Abstract

Lipid nanoparticles (LNPs) can assist in the delivery of nucleic acid inside animal cells, as demonstrated by their use in COVID-19 vaccine development. However, LNPs applicable to bacteria have not been reported. Here, the screening of 511 LNPs containing random combinations of different lipid components identified two LNPs, LNP 496 and LNP 470, that efficiently delivered plasmids into Escherichia coli BW25113. Since Gram-negative bacteria have lipid bilayers, the bacteria are pretreated with LNP-helper that weakens the bacterial membrane. The cationic lipid DOTAP improved delivery of LNP-encapsulated plasmid DNA when present at a molar ratio of 10–25 mol% in the LNP. LNP encapsulation of the Cas13a/gRNA expression vector controlled infection by a clinical Escherichia strain in Galleria mellonela larvae and mouse infection models when used in combination with non-cytotoxic concentrations of polymyxin B, a bacterial membrane disruptor. Together, the results show that LNPs can be useful as a delivery platform for agents that counteract pathogenic bacterial infections.