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...

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Autores principales:	Kevin Xu Zhong, Anna Cho, Christoph M. Deeg, Amy M. Chan, Curtis A. Suttle
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Eukaryotic microbiome 18S rRNA gene Microeukaryote CRISPR-Cas Taxon-specific single-guide RNA gRNA target site Microbial ecology QR100-130
Acceso en línea:	https://doaj.org/article/530c3cf53c4c44d2b55699be120d4ce5
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spelling	oai:doaj.org-article:530c3cf53c4c44d2b55699be120d4ce52021-11-28T12:08:08ZRevealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)10.1186/s40168-021-01180-02049-2618https://doaj.org/article/530c3cf53c4c44d2b55699be120d4ce52021-11-01T00:00:00Zhttps://doi.org/10.1186/s40168-021-01180-0https://doaj.org/toc/2049-2618Abstract 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 AbstractKevin Xu ZhongAnna ChoChristoph M. DeegAmy M. ChanCurtis A. SuttleBMCarticleEukaryotic microbiome18S rRNA geneMicroeukaryoteCRISPR-CasTaxon-specific single-guide RNAgRNA target siteMicrobial ecologyQR100-130ENMicrobiome, Vol 9, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Eukaryotic microbiome 18S rRNA gene Microeukaryote CRISPR-Cas Taxon-specific single-guide RNA gRNA target site Microbial ecology QR100-130
spellingShingle	Eukaryotic microbiome 18S rRNA gene Microeukaryote CRISPR-Cas Taxon-specific single-guide RNA gRNA target site Microbial ecology QR100-130 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)
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
format	article
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
title	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_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_sort	revealing the composition of the eukaryotic microbiome of oyster spat by crispr-cas selective amplicon sequencing (ccsas)
publisher	BMC
publishDate	2021
url	https://doaj.org/article/530c3cf53c4c44d2b55699be120d4ce5
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Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

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