Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)
department or school: Science, Faculty of
department or school: Botany, Department of
department or school: Earth, Ocean and Atmospheric Sciences, Department of
department or school: Microbiology and Immunology, Department of
department or school: Oceans and Fisheries, Institute for the
data provider: DSpace
identifier citation: Microbiome. 2021 Nov 26;9(1):230
rights copyright: The Author(s)
creator: Zhong, Kevin Xu
creator: Cho, Anna
creator: Deeg, Christoph M.
creator: Chan, Amy M.
creator: Suttle, Curtis Arnold, 1955-
issued: 2021-12-01T16:06:56Z
issued: 2021-11-26
description: 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 sequ…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/80297
note: METHODOLOGY Open AccessRevealing the composition of theeukaryotic microbiome of oyster spat byCRISPR-Cas Selective Amplicon Sequencing(CCSAS)Kevin Xu Zhong1*, Anna Cho2, Christoph M. Deeg2, Amy M. Chan1 and Curtis A. Suttle1,2,3,4*AbstractBackground: 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 RNAgene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbesthis approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped bycontaminating 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-guideRNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungalsequences 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 geneamplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used toassess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community ofeukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than othermethods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that werenot detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryoticmicrobiome 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 microbiome…
has type: Article
is shown at: 10.14288/1.0404412
language: eng
peer review status: Reviewed
provider: Vancouver : University of British Columbia Library
publisher: BioMed Central
publisher DOI: 10.1186/s40168-021-01180-0
rights: Attribution 4.0 International (CC BY 4.0)
rights URI: http://creativecommons.org/licenses/by/4.0/
scholar level: Faculty
scholar level: Researcher
subject: Eukaryotic microbiome
subject: 18S rRNA gene
subject: Microeukaryote
subject: CRISPR-Cas
subject: Taxon-specific single-guide RNA
subject: gRNA target site
subject: CasOligo
subject: CCSAS
type: Text
identifier URI: http://hdl.handle.net/2429/80297