Characterization of the eukaryotic microbiome is required to understand the role of microbial communities in health and disease. Such investigation relies on sequencing 18S ribosomal RNA genes (rDNA), which serve as taxonomic markers; however, this is compromised by contaminating host rDNA sequences. To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rDNA sequences of the host. Validation shows that >96.5% of rDNA amplicons from ten model organisms were cleaved, while rDNA from protists and fungi were unaffected. In oyster spat, CCSAS resolved ~8.5-fold more taxa, and several additional major phylogenetic groups when compared to the best available alternative approach. We designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected because of methodological challenges.

中文翻译：

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CRISPR-Cas选择性扩增子测序（CCSAS）揭示后生动物的真核微生物组

要了解微生物群落在健康和疾病中的作用，需要对真核微生物组进行表征。此类研究依赖于对18S核糖体RNA基因（rDNA）进行测序，该基因可作为生物分类标记。但是，这受到污染宿主rDNA序列的影响。为了克服这个问题，我们开发了CRISPR-Cas选择性扩增子测序（CCSAS），这是一种用于鉴定真核微生物组的高分辨率和高效方法。CCSAS使用分类单元特异性单向导RNA（sgRNA）指导Cas9切割宿主的18S rDNA序列。验证显示，来自十个模型生物的rDNA扩增子中> 96.5％都被切割了，而来自原生生物和真菌的rDNA并未受到影响。在牡蛎上，CCSAS解决了大约8.5倍的类群，与最佳的替代方法相比，还有其他几个主要的系统发生群体。我们为约16,000个后生动物和植物类群设计了分类群特异性sgRNA，使CCSAS可广泛用于表征由于方法学难题而被广泛忽略的真核微生物组。