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从人类肠道宏基因组数据中合成一个可共轭系统,用于 Cas9 抗菌药物的靶向投递。

Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials.

机构信息

Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London N6A 5C1, ON, Canada.

Départment de Biologie, Université de Sherbrooke, Sherbrooke J1K 2R1, QC, Canada.

出版信息

ACS Synth Biol. 2023 Dec 15;12(12):3578-3590. doi: 10.1021/acssynbio.3c00319. Epub 2023 Dec 4.

Abstract

Metagenomic sequences represent an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequences requires the identification of suitable candidate systems as starting scaffolds for DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic "trash bin" for genes associated with conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut data set representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugation genes with a differential distribution in human cohorts. We synthesized an entire spp. conjugative system of 54 kb containing at least 47 genes and assembled it into a plasmid, pCitro. We found that pCitro conjugates from to with a 30-fold higher frequency than to , and is compatible with resident plasmids. Mutations in the and conjugation components of pCitro inhibited conjugation. We showed that pCitro can be repurposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and -specific sgRNAs to kill . Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.

摘要

宏基因组序列代表了遗传新颖性的未开发来源,特别是对于可用于基于质粒的 Cas9 衍生抗菌剂递送的可移动系统。然而,仅从宏基因组序列中解锁可移动系统的功能潜力需要鉴定合适的候选系统作为 DNA 合成的起始支架。在这里,我们开发了一种生物信息学方法,该方法可在宏基因组的“垃圾桶”中搜索与通常排除在常见宏基因组分析管道之外的基因座上存在的可移动系统相关的基因。使用代表 2805 个分类学上不同单元的人类宏基因组肠道数据集,我们鉴定了 1598 个包含可移动系统基因的基因座,这些基因座在人类队列中具有不同的分布。我们合成了一个完整的 spp.可移动系统,大小为 54kb,包含至少 47 个基因,并将其组装到质粒 pCitro 中。我们发现 pCitro 从 到 的转导频率比到 的高 30 倍,并且与 中的常驻质粒兼容。pCitro 中的 和 可移动系统组件的突变抑制了转导。我们表明,通过用 TevCas9 核酸酶和 -特异性 sgRNA 编程 pCitro,可以将其重新用作抗菌剂递送剂,以杀死 。我们的研究揭示了宏基因组数据中大量未表征的可移动系统,并描述了一种实验框架,可将这些大型遗传系统作为新型靶向适应性 Cas9 编辑的细菌基因组的递送载体进行激活。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ede8/10729033/7663e8ca79ea/sb3c00319_0001.jpg

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