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Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants.

Naiyaphat Nittayasut·Teerapong Yata·Sunisa Chirakul·Navapon Techakriengkrai·Pattrarat Chanchaithong
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Abstract

Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.

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CRISPR-Cas SystemsEscherichia coliBacteriophagesbeta-LactamasesEscherichia coli ProteinsPlasmidsPromoter Regions, GeneticGene EditingAnti-Bacterial AgentsJournal ArticleResearch Support, Non-U.S. Gov't

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Naiyaphat Nittayasut, Teerapong Yata, Sunisa Chirakul, Navapon Techakriengkrai, Pattrarat Chanchaithong. 2024-05-16. Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants.. https://doi.org/10.1371/journal.pone.0303555

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