Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)
Abstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic commun...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-11-01
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| Series: | Microbiome |
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| Online Access: | https://doi.org/10.1186/s40168-021-01180-0 |
| _version_ | 1818434222112636928 |
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| author | Kevin Xu Zhong Anna Cho Christoph M. Deeg Amy M. Chan Curtis A. Suttle |
| author_facet | Kevin Xu Zhong Anna Cho Christoph M. Deeg Amy M. Chan Curtis A. Suttle |
| author_sort | Kevin Xu Zhong |
| collection | DOAJ |
| description | Abstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. Results 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 rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, 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. Conclusion CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Video Abstract |
| first_indexed | 2024-12-14T16:33:33Z |
| format | Article |
| id | doaj.art-530c3cf53c4c44d2b55699be120d4ce5 |
| institution | Directory Open Access Journal |
| issn | 2049-2618 |
| language | English |
| last_indexed | 2024-12-14T16:33:33Z |
| publishDate | 2021-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbiome |
| spelling | doaj.art-530c3cf53c4c44d2b55699be120d4ce52022-12-21T22:54:31ZengBMCMicrobiome2049-26182021-11-019111710.1186/s40168-021-01180-0Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)Kevin Xu Zhong0Anna Cho1Christoph M. Deeg2Amy M. Chan3Curtis A. Suttle4Department of Earth, Ocean, and Atmospheric Sciences, The University of British ColumbiaDepartment of Microbiology and Immunology, The University of British ColumbiaDepartment of Microbiology and Immunology, The University of British ColumbiaDepartment of Earth, Ocean, and Atmospheric Sciences, The University of British ColumbiaDepartment of Earth, Ocean, and Atmospheric Sciences, The University of British ColumbiaAbstract Background The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. Results 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 rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, 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. Conclusion CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Video Abstracthttps://doi.org/10.1186/s40168-021-01180-0Eukaryotic microbiome18S rRNA geneMicroeukaryoteCRISPR-CasTaxon-specific single-guide RNAgRNA target site |
| spellingShingle | Kevin Xu Zhong Anna Cho Christoph M. Deeg Amy M. Chan Curtis A. Suttle Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS) Microbiome Eukaryotic microbiome 18S rRNA gene Microeukaryote CRISPR-Cas Taxon-specific single-guide RNA gRNA target site |
| title | Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS) |
| title_full | Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS) |
| title_fullStr | Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS) |
| title_full_unstemmed | Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS) |
| title_short | Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS) |
| title_sort | revealing the composition of the eukaryotic microbiome of oyster spat by crispr cas selective amplicon sequencing ccsas |
| topic | Eukaryotic microbiome 18S rRNA gene Microeukaryote CRISPR-Cas Taxon-specific single-guide RNA gRNA target site |
| url | https://doi.org/10.1186/s40168-021-01180-0 |
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