Targeted-Antibacterial-Plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity

Abstract

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on Targeted-Antibacterial-Plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step towards the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.

High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing

“…Similar results were obtained when the COP strain was introduced as a prophylactic treatment 12 h prior to the gavage of the target and non‐target strains, leading to a specific ˜30‐fold depletion of the targeted KN02 strain (median = 96.4% decrease 36 h after the administration of the COP) (Fig EV2). As previously reported (Reuter et al , 2021), escaper mutants, which are target bacteria that received the CRISPR module but were not eliminated, can be detected at low frequencies during in vitro (Appendix Fig S2A) and in situ (Appendix Fig S2B) experiments. Sequencing these escapers revealed deleterious mutations of various types in either the gRNA or cas9 gene (Appendix Fig S1C and D).…”

“…However, the impact of the COP strategy on the microbiota might differ depending on the selected gRNA sequences. Several groups have developed spacer design algorithms to maximize on‐target activity and help limit off‐targeting (Guo et al , 2018; Calvo‐Villamañán et al , 2020; Reuter et al , 2021). By adapting these strategies to consider the entire composition of the microbiome, one could in principle design gRNA to specifically eliminate specific bacterial strains or species from the microbiota.…”

Tailoring CRISPR-Cas Immunity for the Degradation of Antibiotic Resistance Genes

“…Conjugation is another possible way for delivery of genetic systems, as it has a broad spectrum of recipient cells, and the feasibility has been demonstrated in principle. [23][24][25] By applying conjugation machinery, when the donor cell with VADER contacts with the recipient cell harboring ARG, it delivers VADER and then VADER will degrade the target ARG in vivo (Figure 4A).…”

“…Therefore, by applying CRISPR-Cas immunity, ARGs will be degraded. Pioneer studies have validated that CRISPR-Cas system can eliminate chromosomal ARGs for medical applications, [21][22][23][24] and the system can be delivered via conjugation. 24 These advances, however, have difficulties in finding a practical scenario.…”

“…Pioneer studies have validated that CRISPR-Cas system can eliminate chromosomal ARGs for medical applications, [21][22][23][24] and the system can be delivered via conjugation. 24 These advances, however, have difficulties in finding a practical scenario. The bottlenecks mainly lie in delivery, as (1) conjugation needs physical contact between donor and recipient cells, and (2) a threshold mount of bacterial cells and contacting time is necessary.…”