High-throughput sequencing of bacterial 16S rRNA gene (16S-seq) is a useful and common method for studying bacterial community structures. However, contamination of the 16S rRNA genes from the mitochondrion and plastid hinders the sensitive bacterial 16S-seq in plant microbiota profiling, especially for some plant species such as rice. To date, efficiently mitigating such host contamination without a bias is challenging in 16S rRNA gene-based amplicon sequencing. We developed Cas-16S-seq method to reduce abundant host contamination for plant microbiota profiling. This method utilizes the Cas9 nuclease and specific guide RNA (gRNA) to cut 16S rRNA targets during library construction, thereby removing host contamination in 16S-seq. We used rice as an example to validate the feasibility and effectiveness of Cas-16S-seq. We established a bioinformatics pipeline to design gRNAs that specifically target rice 16S rRNA genes without bacterial 16S rRNA off-targets. We compared the effectiveness of Cas-16S-seq with that of the commonly used 16S-seq method for artificially mixed 16S rRNA gene communities, paddy soil, rice root, and phyllosphere samples. The results showed that Cas-16S-seq substantially reduces the fraction of rice 16S rRNA gene sequences from 63.2 to 2.9% in root samples and from 99.4 to 11.6% in phyllosphere samples on average. Consequently, Cas-16S-seq detected more bacterial species than the 16S-seq in plant samples. Importantly, when analyzing soil samples, Cas-16S-seq and 16S-seq showed almost identical bacterial communities, suggesting that Cas-16S-seq with host-specific gRNAs that we designed has no off-target in rice microbiota profiling. Our Cas-16S-seq can efficiently remove abundant host contamination without a bias for 16S rRNA gene-based amplicon sequencing, thereby enabling deeper bacterial community profiling with a low cost and high flexibility. Thus, we anticipate that this method would be a useful tool for plant microbiomics.

中文翻译：

---

工程CRISPR / Cas9可减轻基于16S rRNA基因的扩增子测序的大量宿主污染。

细菌16S rRNA基因（16S-seq）的高通量测序是研究细菌群落结构的有用且常用的方法。但是，线粒体和质体中的16S rRNA基因受到污染，阻碍了植物微生物区系分析中敏感细菌16S-seq的产生，特别是对于某些植物物种，例如水稻。迄今为止，在基于16S rRNA基因的扩增子测序中，有效地缓解宿主污染而无偏倚是一项挑战。我们开发了Cas-16S-seq方法，以减少植物微生物群图分析中大量宿主污染。该方法利用Cas9核酸酶和特定的指导RNA（gRNA）在文库构建过程中切割16S rRNA靶标，从而消除了16S-seq中的宿主污染。我们以大米为例，验证了Cas-16S-seq的可行性和有效性。我们建立了一条生物信息学流水线，以设计专门针对水稻16S rRNA基因的gRNA，而不会使细菌16S rRNA脱靶。我们比较了Cas-16S-seq与通常使用的16S-seq方法对人工混合的16S rRNA基因群落，水稻土，稻根和叶根样品的有效性。结果表明，Cas-16S-seq显着降低了水稻16S rRNA基因序列在根样品中的比例，从63.2％降至2.9％，在叶球样品中平均从99.4％降至11.6％。因此，Cas-16S-seq在植物样品中检测到的细菌种类比16S-seq多。重要的是，在分析土壤样品时，Cas-16S-seq和16S-seq显示几乎相同的细菌群落，这表明我们设计的具有宿主特异性gRNA的Cas-16S-seq在水稻微生物群谱分析中没有目标。我们的Cas-16S-seq可以有效去除大量宿主污染，而不会偏向基于16S rRNA基因的扩增子测序，从而以低成本和高灵活性实现更深的细菌群落分析。因此，我们预期该方法将是用于植物微生物学的有用工具。