Genetic Manipulation of a CAST of Characters in a Microbial Community.
Genetic Manipulation of a CAST of Characters in a Microbial Community.
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微生物群落中一系列特征的遗传操作。
DOI：
10.1089/crispr.2022.29142.dmo
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Bondy-Denomy,Joseph
中科院分区：
文献类型：
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作者：
Mozumdar,Deepto;Csörgő,Bálint;Bondy-Denomy,Joseph
地球上几乎所有的微生物都生活在群落中。然而，大多数实验研究使用单一培养来阐明基因和微生物的功能。迄今为止，修改微生物群落中单个组分的DNA并在接近天然的环境中调查这些操作的表型后果一直具有挑战性。大多数微生物尚未在其原生环境之外被分离或成功培养。即使对于那些可以培养的细菌，我们也往往缺乏基因编辑所必需的强大工具（即复制质粒）。因此，对于解决这些众多挑战并促进我们对天然微生物群落中存在的复杂现象的理解的工具存在着未满足的需求。1为了解决这些问题，CRISPR的两位先驱Jennifer Doudna和Jillian Banfield领导了一个多学科团队（包括CRISPR杂志的主编Rodolphe Bengeou），该团队在《自然微生物学》上报道了生物信息学和遗传工具包的开发，以识别和操纵社区中的可编辑微生物。2在这个工作流程中，第一步涉及对细菌群落进行预筛选，以识别适合核酸递送和基因组编辑的特定细菌。这是通过称为环境转化测序（ET-Seq;图1）的过程实现的。在这个管道中，感兴趣的复杂微生物群落首先暴露于通过三种不同方法递送的随机整合的mariner转座子：缀合，电穿孔和自然转化。随后，在不存在任何选择的情况下，提取总群落DNA并测序以评估每个生物体中基因组整合的位置和频率。使用生物信息学管道将插入事件的频率标准化为内部标准（添加到微生物样品中）并解释单个生物体的宏基因组丰度，作者能够定量测量每个社区成员的转座子插入的物种特异性百分比。因此，ET-Seq能够以定量的方式评估群落中每种细菌物种对遗传操作的相对适应性。在他们的研究中，作者测试了ET-Seq（1）由来自三个独立门的九种不同微生物组成的合成土壤群落和（2）离体培养的婴儿肠道微生物样本。使用ET-Seq方法，作者能够在两个样品中重复检测基因插入，鉴定样品中适合外源DNA整合的微生物，并确定递送基因编辑货物的最佳方法。值得注意的是，没有检测到将外源DNA引入到包括群落中较稀有物种的多种微生物中。ET-Seq管道的优雅在于提供快速筛选微生物群落的遗传可及性的能力，而无需培养和测试组成微生物的单个菌株。在用ET-Seq鉴定了用于基因编辑的候选微生物后，Rubin等人设计了一种CRISPR相关的Tn 7转座子（CAST）系统，以将靶向基因编辑货物递送给这些细菌。CAST系统（也称为通过引导RNA辅助靶向插入转座因子的整合）首先从细菌和古细菌基因组的生物信息学筛选中鉴定为天然存在的
Nearly all microbes on the planet live in communities. Most experimental studies, however, use monoculture to elucidate gene and microbe functions. Modifying the DNA of individual constituents in a microbial community and investigating the phenotypic consequences of these manipulations in a near-native context has thus far been challenging. Most microbes have not been isolated or successfully cultured outside their native environments. Even for those bacteria that can be cultivated, we often lack the robust tools (ie, replicating plasmids) necessary for gene editing. Thus, there is an unmet need for tools to tackle these numerous challenges and to advance our understanding of the complex phenomena present in native microbial communities. 1 To address these problems, two CRISPR pioneers, Jennifer Doudna and Jillian Banfield, have lead a multidisciplinary team (including The CRISPR Journal’s Editor-in-Chief, Rodolphe Barrangou) that reports in Nature Microbiology the development of a bioinformatic and genetic toolkit to identify and manipulate the editable microbes in a community. 2 In this workflow, the first step involves prescreening a bacterial community to identify specific bacteria that are amenable to the delivery of nucleic acids and genome editing. This is achieved through a process called environmental transformation sequencing (ET-Seq; Fig. 1). In this pipeline, the complex microbial community of interest is first exposed to a randomly integrating mariner transposon delivered via three separate methods: conjugation, electroporation, and natural transformation. Subsequently, in the absence of any selection, the total community DNA is extracted and sequenced to evaluate the location and frequency of genomic integration in each organism. Using a bioinformatic pipeline that normalizes the frequency of the insertion events to an internal standard (added to the microbial sample) and accounts for metagenomic abundance of the individual organisms, the authors were able to measure the species-specific percentage of transposon insertion of each community member quantitatively. ET-Seq is therefore able to evaluate the relative amenability of each bacteria species agnostically within the community to genetic manipulation in a quantitative fashion.In their study, the authors tested ET-Seq on (1) a synthetic soil community consisting of nine distinct microbes from three separate phyla and (2) an infant gut microbe sample cultured ex vivo. Using the ET-Seq approach, the authors were able to detect genetic insertions reproducibly in both samples, identify microbes within the samples that were amenable to foreign DNA integration, and determine the best method for delivery of the gene editing cargo. Notably, introduction of foreign DNA into multiple microbes comprising the rarer species in the community was not detected. The elegance of the ET-Seq pipeline lies in providing the ability to screen a microbial community rapidly for genetic accessibility without the need for culturing and testing individual strains of the constituent microbes. Having identified candidate microbes with ET-Seq for gene editing, Rubin et al. engineered a CRISPR-associated Tn7 transposon (CAST) system to deliver targeted gene editing cargo to these bacteria. CAST systems (also called INTEGRATE-insertion of transposable elements by guide RNA–assisted targeting) were first identified from a bioinformatic screen of bacterial and archaeal genomes3 as a naturally occurring