A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness.

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen (i.e., COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at day zero, i.e., the time of the first reported case, would be of significant value. Next-generation sequencing (NGS) has such capabilities; however, it has limited detection sensitivity for low-copy-number pathogens. Here, we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS work flow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

Morphological and transcriptional effects of crude oil and dispersant exposure on the marine sponge Cinachyrella alloclada.

Marine sponges play important roles in benthic ecosystems. More than providing shelter and food to other species, they help maintain water quality by regulating nitrogen and ammonium levels in the water, and bioaccumulate heavy metals. This system, however, is particularly sensitive to sudden environmental changes including catastrophic pollution event such as oil spills. Hundreds of oil platforms are currently actively extracting oil and gas in the Gulf of Mexico. To test the vulnerability of the benthic ecosystems to oil spills, we utilized the Caribbean reef sponge, Cinachyrella alloclada, as a novel experimental indicator. We have exposed organisms to crude oil and oil dispersant for up to 24 h and measured resultant gene expression changes. Our findings indicate that 1-hour exposure to water accommodated fractions (WAF) was enough to elicit massive shifts in gene expression in sponges and host bacterial communities (8052 differentially expressed transcripts) with the up-regulation of stress related pathways, cancer related pathways, and cell integrity pathways. Genes that were upregulated included heat shock proteins, apoptosis, oncogenes (Rab/Ras, Src, CMYC), and several E3 ubiquitin ligases. 24-hour exposure of chemically enhanced WAF (CE-WAF) had the greatest impact to benthic communities, resulting in mostly downregulation of gene expression (4248 differentially expressed transcripts). Gene deregulation from 1-hour treatments follow this decreasing trend of toxicity: WAF > CE-WAF > Dispersant, while the 24-hour treatment showed a shift to CE-WAF > Dispersant > WAF in our experiments. Thus, this study supports the development of Cinachyrella alloclada as a research model organism and bioindicator species for Florida reefs and underscores the importance of developing more efficient and safer ways to remove oil in the event of a spill catastrophe.

Holo-Transcriptome Sequences From the Tropical Marine Sponge Cinachyrella alloclada.

Marine sponge transcriptomes are underrepresented in current databases. Furthermore, only 2 sponge genomes are available for comparative studies. Here we present the assembled and annotated holo-transcriptome of the common Florida reef sponge from the species Cinachyrella alloclada. After Illumina high-throughput sequencing, the data assembled using Trinity v2.5 confirmed a highly symbiotic organism, with the complexity of high microbial abundance sponges. This dataset is enriched in poly-A selected eukaryotic, rather than microbial transcripts. Overall, 39 813 transcripts with verified sponge sequence homology coded for 8496 unique proteins. The average sequence length was found to be 946 bp with an N50 sequence length of 1290 bp. Overall, the sponge assembly resulted in a GC content of 51.04%, which is within the range of GC bases in a eukaryotic transcriptome. BUSCO scored completeness analysis revealed a completeness of 60.3% and 60.1% based on the Eukaryota and Metazoa databases, respectively. Overall, this study points to an overarching goal of developing the C. alloclada sponge as a useful new experimental model organism.