Re-sensitization of Mycobacterium smegmatis to Rifampicin Using CRISPR Interference Demonstrates Its Utility for the Study of Non-essential Drug Resistance Traits.
Re-sensitization of Mycobacterium smegmatis to Rifampicin Using CRISPR Interference Demonstrates Its Utility for the Study of Non-essential Drug Resistance Traits.
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使用 CRISPR 干扰使耻垢分枝杆菌对利福平重新敏感，证明了其在非必需耐药性状研究中的实用性。
DOI：
10.3389/fmicb.2020.619427
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发表时间：
2020
影响因子：
5.2
通讯作者：
Kendall SL
中科院分区：
文献类型：
--
作者：
Faulkner V;Cox AA;Goh S;van Bohemen A;Gibson AJ;Liebster O;Wren BW;Willcocks S;Kendall SL
更好地了解结核分枝杆菌（Mtb）中涉及抗生素耐药性的基因对于设计改进的治疗方法是必要的。聚类规则间隔短回文重复干扰（CRISPRi）先前已被用于分枝杆菌中，通过证明基因的必要性来识别新的药物靶点。这里提出的工作表明，它也可以有效地应用于研究非必需基因参与抗生素耐药性。利用CRISPRi沉默了与利福平耐药有关的adp -核糖基转移酶（Arr）的表达，并测定了其对利福平敏感性的影响。基因沉默导致最低抑制浓度（MIC）下降，类似于先前报道的arr缺失突变体。文献中关于化脓性链球菌dCas9 （dCas9Spy）的毒性存在相互矛盾的证据。在本研究中，dCas9Spy的表达对耻垢分枝杆菌的生存能力没有影响。aTc诱导剂的浓度低于先前描述的浓度，诱导时间缩短，从而实现了沉默。最后，设计靶向转录起始或延伸早期阶段的小向导rna （sgRNAs）对利福平敏感性影响最大。本研究表明，在非必需基因的研究中，基于CRISPRi的基因沉默可以与基因缺失一样有效，并进一步有助于CRISPRi的sgrna的设计和诱导。这种方法可以应用于其他非必需的抗菌素耐药基因，如药物外排泵。
A greater understanding of the genes involved in antibiotic resistance in Mycobacterium tuberculosis (Mtb) is necessary for the design of improved therapies. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been previously utilized in mycobacteria to identify novel drug targets by the demonstration of gene essentiality. The work presented here shows that it can also be usefully applied to the study of non-essential genes involved in antibiotic resistance. The expression of an ADP-ribosyltransferase (Arr) involved in rifampicin resistance in Mycobacterium smegmatis was silenced using CRISPRi and the impact on rifampicin susceptibility was measured. Gene silencing resulted in a decrease in the minimum inhibitory concentration (MIC) similar to that previously reported in an arr deletion mutant. There is contradictory evidence for the toxicity of Streptococcus pyogenes dCas9 (dCas9Spy) in the literature. In this study the expression of dCas9Spy in M. smegmatis showed no impact on viability. Silencing was achieved with concentrations of the aTc inducer lower than previously described and with shorter induction times. Finally, designing small guide RNAs (sgRNAs) that target transcription initiation, or the early stages of elongation had the most impact on rifampicin susceptibility. This study demonstrates that CRISPRi based gene silencing can be as impactful as gene deletion for the study of non-essential genes and further contributes to the knowledge on the design and induction of sgRNAs for CRISPRi. This approach can be applied to other non-essential antimicrobial resistance genes such as drug efflux pumps.
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影响因子：
9.8
作者：
Ayhan DH;Tamer YT;Akbar M;Bailey SM;Wong M;Daly SM;Greenberg DE;Toprak E
通讯作者：
Toprak E
影响因子：
3.7
作者：
Agrawal, Priyanka;Miryala, Sandeep;Varshney, Umesh
通讯作者：
Varshney, Umesh
影响因子：
4.9
作者：
McNeil, Matthew B.;Cook, Gregory M.
通讯作者：
Cook, Gregory M.
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
8.8
作者：
Cortes T;Schubert OT;Rose G;Arnvig KB;Comas I;Aebersold R;Young DB
通讯作者：
Young DB