A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

Abstract

The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.

Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens

“…As a result, the vast majority of pathogens will die with only a small fraction surviving by mutating the AR gene. Therefore, CRISPR antimicrobials can specifically eradicate AR pathogens in a complex bacterial community or cure their antibiotic resistance, and have been designed for target elimination or re-sensitization of different MDR pathogens, like Escherichia coli 3 , 10 , 12 – 14 , Staphylococcus aureus 4 , Clostridioides difficile 11 , and Enterococcus faecalis 15 .…”

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool

“…Several previous studies aimed to resensitize bacteria by conjugatively delivering an engineered CRISPR plasmid [10,11,[20][21][22][23], but most of these deployed a mobilizable CRISPR delivery tool that requires either a second conjugative plasmid or an engineered donor strain [11,20,[22][23][24]. Crucially, the application of a CRISPR delivery tool has been shown to be more effective when conjugative machinery and CRISPR machinery are encoded on the same genetic element (in cis), rather than on separate plasmids (in trans) [25].…”

“…Beyond this, target plasmid removal efficiency by CRISPR delivery tools could depend on other factors, such as target plasmid mobility [ 11 ], plasmid copy number [ 33 ], or the presence of other payload genes on target plasmids or in target genomes such as anti-CRISPR proteins [ 34 ] or toxin–antitoxin systems. These should be further experimentally investigated for optimization of AMR plasmid removal using CRISPR-Cas9.…”

“…CRISPR delivery tools have been engineered using various genetic backbones, for instance non-replicating phage plasmids (phagemids) [7,8], expression vectors [9], or synthetic conjugative or mobilizable plasmids [10,11]. These are effective at blocking transfer of resistance genes into specific strains and can remove AMR genes from them.…”

15 years of microbial biotechnology: the time has come to think big—and act soon

“…Ongoing, mostly academic efforts to cope with the problem involve surveying of peptide antibiotics (sometimes found in the most unexpected places; Torres et al ., 2021), new functional tests for molecules with antimicrobial activity (Wrighton, 2018) or reliance on phages (Gordillo Altamirano and Barr, 2019). Other propositions for dealing with new pathogens and antibiotic resistances involve surveillance strains in the microbiome that conjugatively deliver plasmids able to activate host‐killing circuits when a virulence signal or a resistance gene is detected in a recipient bacterium (López‐Igual et al ., 2019; Wongpayak et al ., 2021). Whether such smart strategies will make it to actual patients remains an open question, but innovative ideas of this sort are badly needed and will surely emerge with more frequency in the foreseeable future.…”