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 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.

A CRISPR‐based strategy for targeted sequencing in biodiversity science

“…For example, DNA extraction methods (Kang et al., 2023; Russo et al., 2022), the number of purification steps used during library preparation (De La Cerda et al., 2023), the specific Cas system deployed (e.g., Cas9 vs. Cas3 or Cas12a [Schultzhaus et al., 2021]), and the sequencing platform utilized (e.g., Oxford Nanopore vs. Illumina or PacBio [Li & Harkess, 2018]) may need to be optimized in order to ensure adequate on‐target sequence coverage. There are encouraging strategies to deplete non‐target sequences, such as in host DNA in microbiome studies, using CRISPR‐Cas selective amplicon sequencing (Zhong et al., 2021). Strategies to enhance the enrichment of on‐target reads also include methods for tiling gRNAs, whereby overlapping gRNAs can be used to extend the enrichment of the target region (López‐Girona et al., 2020), or to improve the median depth of coverage for a particular locus (Gilpatrick et al., 2020).…”

A CRISPR-based strategy for targeted sequencing in biodiversity science

“…Encouraging strategies to enhance enrichment of on-target reads include methods for tiling gRNAs, whereby overlapping gRNAs can be used to extend the enrichment of the target region (López-Girona et al, 2020), or to improve the median depth of coverage for a particular locus (Gilpatrick et al, 2020). In addition, there are encouraging strategies to deplete non-target sequences, such as host DNA in microbiome studies, using CRISPR-Cas selective amplicon sequencing (Zhong et al, 2021).…”

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

“…CRISPRclean has been used in microbiome analyses (Uranga et al, 2022), single cell transcriptomics (Pandey et al, 2023), and ribosomal RNA (rRNA) depletion (Cerón et al, 2022). Other CRISPR-Cas based depletion methods have also been used in immunotherapy for cancer treatment by down-regulating the methionine transporter SLC43A2 and restricting the methionine uptake by tumor cells (Huang et al, 2023), used as an antimicrobial agent by selectively depleting antibiotic resistant strains of bacteria (Rodrigues et al, 2019), or used to deplete eukaryotic host DNA for amplicon sequencing in microbiome studies (Zhong et al, 2021). The diverse range of demonstrated applications of DASH and other CRISPR-Cas technologies provide an encouraging foundation for its potential use in eDNA monitoring of aquatic environments.…”