CRISPR-Cas9 介导的耐甲氧西林金黄色葡萄球菌多药耐药性基因靶向。

IF 3.7 4区生物学 Q2 GENETICS & HEREDITY

CRISPR Journal Pub Date : 2024-11-08 DOI:10.1089/crispr.2024.0001

Aysegul Ates, Cihan Tastan, Safak Ermertcan

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引用次数: 0

摘要

抗生素耐药性是一个全球性的健康危机，限制了现有治疗药物的疗效。我们针对甲氧西林（mecA）、庆大霉素（aacA）和环丙沙星（grlA、grlB）耐药基因，探索了基于CRISPR-Cas的抗菌药物，以对抗耐甲氧西林金黄色葡萄球菌（MRSA）的多重耐药性。将带有特定单导 RNA 的 CRISPR 质粒电穿孔到 MRSA 菌株中。实时聚合酶链反应评估了基因表达的变化，而抗生素药敏试验（AST）则评估了耐药性状况。结果显示，mecA 表达量减少了 1.5 倍，grlA 减少了 5.5 倍，grlB 减少了 6 倍，aacA 表达量减少了 4 倍。ASTs 表明对 beta-内酰胺类、喹诺酮类和氨基糖苷类抗生素的耐药性发生了逆转。Western 印迹分析显示，青霉素结合蛋白 2a 的表达量减少了 70%。桑格测序证实了 grlB 和 aacA 基因的点突变。我们的研究结果凸显了 CRISPR-Cas9 技术在恢复抗生素对耐多药病原体疗效方面的潜力。

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CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus.

Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.

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来源期刊

CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology

CiteScore

6.30

自引率

2.70%

发文量

期刊介绍： In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.