Revealing the Composition of the Eukaryotic Microbiome of Oyster Spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

Abstract

BackgroundThe 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. ResultsTo 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. ConclusionCCSAS 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. Keywords: Eukaryotic microbiome, 18S rRNA gene, Microeukaryote, CRISPR-Cas, Taxon-specific single-guide RNA, gRNA-target-site, CasOligo, CCSAS

Elongation arrest blocking primers enhance parasite detection in 18S rRNA metabarcoding study of Mesonauta festivus gut eukaryotic communities.

“…Previous studies reached near complete inhibition of host target gene amplification using both annealing blocking primers and CCSAS (Vestheim and Jarman 2008, Liu et al . 2019, Zhong et al . 2021.…”

“…2021. Furthermore, Zhong et al (2021) already developed guide RNAs for 16 000 referenced eukaryotes. Their method could also be implemented for unreferenced organisms using sanger sequencing, as we did in this study.…”

“…There are multiple methods to reduce the quantity of host DNA amplicons in samples. Recently, Zhong et al . (2021) developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS) based on taxon-specific single-guide RNA to direct Cas9 to cut host 18S rRNA gene sequences.…”

“…2021, the usage of blocking primers to describe host-parasite interactions could still benefit from additional testing of the method for other non-referenced species. Moreover, additional data should help compare this method to the newly developed approach based on CRISPR-Cas (Zhong et al . 2021).…”

Microscopic marine invertebrates are reservoirs for cryptic and diverse protists and fungi

“…The co-amplification of host DNA when targeting symbiotic microbial eukaryotes can often dwarf non-metazoan reads and nullify attempts to fully characterize the eukaryome. However, various approaches have been developed to mitigate this problem, and sequence characterization of the eukaryotic microbiome is a possibility [8,9].…”