Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing
Open Access
- 3 June 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Microbiome
- Vol. 8 (1), 1-15
- https://doi.org/10.1186/s40168-020-00859-0
Abstract
Background: 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. Results: 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. Conclusion: 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.Keywords
Funding Information
- National Natural Science Foundation of China (31622047)
- National Transgenic Science and Technology Program (2018ZX08010-05B)
- National Key Laboratory of Crop Genetic Improvement
This publication has 57 references indexed in Scilit:
- phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census DataPLOS ONE, 2013
- Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteriaProceedings of the National Academy of Sciences of the United States of America, 2012
- Defining the core Arabidopsis thaliana root microbiomeNature, 2012
- Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiotaNature, 2012
- Structure, function and diversity of the healthy human microbiomeNature, 2012
- Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platformsThe ISME Journal, 2012
- An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaeaThe ISME Journal, 2011
- UCHIME improves sensitivity and speed of chimera detectionBioinformatics, 2011
- Global patterns of 16S rRNA diversity at a depth of millions of sequences per sampleProceedings of the National Academy of Sciences of the United States of America, 2010
- A renaissance for the pioneering 16S rRNA geneCurrent Opinion in Microbiology, 2008