CRISPR interference for blaNDM-1 and blaOXA-48 genes suppression in carbapenemase-resistant Klebsiella pneumoniae strains

IF 1 Q4 GENETICS & HEREDITY

Gene Reports Pub Date : 2025-03-28 DOI:10.1016/j.genrep.2025.102210

Yeganeh Hajizadeh , Mana Oloomi

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Abstract

One treatment option for multidrug-resistant strains, such as carbapenemase-producing Klebsiella pneumoniae, is the CRISPR interference (CRISPRi) technique, developed to address issues caused by these pathogens. This system includes a gRNA and a dCas9. This study investigates the effectiveness of the CRISPRi system in reducing meropenem resistance in K. pneumoniae harboring β-lactamase strains, specifically the bla_NDM-1 and bla_OXA-48 genes, both separately and simultaneously, by optimizing sgRNA designs. We designed and incorporated two optimal sgRNAs, which were then cloned into a pJMP1363 plasmid with the CRISPRi system to target the expression of the bla_NDM-1 and bla_OXA-48 genes separately and simultaneously. Antimicrobial resistance was evaluated using the MIC test, and gene expression was measured with qRT-PCR. The MIC test results showed a decrease in meropenem resistance in K. pneumoniae strains carrying the pJMP1363 construct (sgRNA+). The qRT-PCR analysis indicated a reduction in mRNA expression for strains carrying the construct (sgRNA+), with a 67-fold and 31-fold decrease for bla_OXA-48 and bla_NDM-1, respectively, and a 100-fold decrease for strains with both bla_OXA-48 and bla_NDM-1. The decrease in meropenem resistance was more noticeable in strains harboring both resistance genes compared to strains with either gene alone. The study also demonstrated that both sgRNA_OXA-48 and sgRNA_NDM-1, targeting the non-template and template strands of the respective genes, effectively lowered transcription levels of the target genes. Notably, sgRNA_OXA-48 significantly reduced the expression of the bla_OXA-48 gene.

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CRISPR干扰对碳青霉烯酶耐药肺炎克雷伯菌blaNDM-1和blaOXA-48基因的抑制

针对多药耐药菌株（如产生碳青霉烯酶的肺炎克雷伯菌）的一种治疗选择是CRISPR干扰（CRISPRi）技术，该技术是为了解决这些病原体引起的问题而开发的。该系统包括一个gRNA和一个dCas9。本研究通过优化sgRNA设计，考察了CRISPRi系统分别或同时降低携带β-内酰胺酶菌株（特别是blaNDM-1和blaOXA-48基因）的肺炎克雷伯菌对美罗培南耐药性的有效性。我们设计并整合了两个最佳的sgrna，然后用CRISPRi系统将其克隆到pJMP1363质粒中，分别和同时靶向blaNDM-1和blaOXA-48基因的表达。采用MIC试验评估耐药性，采用qRT-PCR检测基因表达。MIC试验结果显示携带pJMP1363构建体（sgRNA+）的肺炎克雷伯菌对美罗培南的耐药性降低。qRT-PCR分析显示，携带该构建体（sgRNA+）的菌株mRNA表达量降低，blaOXA-48和blaNDM-1分别降低67倍和31倍，blaOXA-48和blaNDM-1同时携带的菌株mRNA表达量降低100倍。与单独携带任何一种基因的菌株相比，携带两种耐药基因的菌株对美罗培南的耐药性下降更为明显。该研究还表明，sgRNAOXA-48和sgRNANDM-1分别靶向各自基因的非模板链和模板链，有效降低了靶基因的转录水平。值得注意的是，sgRNAOXA-48显著降低了blaOXA-48基因的表达。

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来源期刊

Gene Reports Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

3.30

自引率

7.70%

发文量

246

审稿时长

49 days

期刊介绍： Gene Reports publishes papers that focus on the regulation, expression, function and evolution of genes in all biological contexts, including all prokaryotic and eukaryotic organisms, as well as viruses. Gene Reports strives to be a very diverse journal and topics in all fields will be considered for publication. Although not limited to the following, some general topics include: DNA Organization, Replication & Evolution -Focus on genomic DNA (chromosomal organization, comparative genomics, DNA replication, DNA repair, mobile DNA, mitochondrial DNA, chloroplast DNA). Expression & Function - Focus on functional RNAs (microRNAs, tRNAs, rRNAs, mRNA splicing, alternative polyadenylation) Regulation - Focus on processes that mediate gene-read out (epigenetics, chromatin, histone code, transcription, translation, protein degradation). Cell Signaling - Focus on mechanisms that control information flow into the nucleus to control gene expression (kinase and phosphatase pathways controlled by extra-cellular ligands, Wnt, Notch, TGFbeta/BMPs, FGFs, IGFs etc.) Profiling of gene expression and genetic variation - Focus on high throughput approaches (e.g., DeepSeq, ChIP-Seq, Affymetrix microarrays, proteomics) that define gene regulatory circuitry, molecular pathways and protein/protein networks. Genetics - Focus on development in model organisms (e.g., mouse, frog, fruit fly, worm), human genetic variation, population genetics, as well as agricultural and veterinary genetics. Molecular Pathology & Regenerative Medicine - Focus on the deregulation of molecular processes in human diseases and mechanisms supporting regeneration of tissues through pluripotent or multipotent stem cells.