Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases

Hypoxia stress exerts responses involving the HPA and HPT axes.

<p>Under hypoxia, the potential neuro-endocrine-immune/metabolism networks contribute to the regulation of moderate inflammation and the maintenance of energy balance. Hypoxia may inhibit the hypothalamic-pituitary-adrenal (HPA) axis and induce the expression of ET-1/ADM, and the latter then promote the expression of IL-6/TNF-α and form a positive feedback loop with them. On the other hand, the ET-1/ADM and IL-6/TNF-α may in turn activate the HPA axis, and the latter subsequently induces glucocorticoids and generates a negative feedback to inhibit <i>ET-1/ADM</i> and <i>IL-6/TNF-α</i> expression to reduce the inflammatory responses in brain. Therefore a HPA axis-ET-1/ADM-IL-6/TNF-α feedback regulatory loop is outlined here. Besides, both SOCS-1 and SOCS-3 may have complementary roles in down-regulating <i>IL-6</i> and <i>TNF-α</i>, and both IL-6 and TNF-α have reciprocal functions to induce <i>SOCS-1</i> and <i>SOCS-3</i> expression, thus constituting a SOCS-1/3-dependent feedback regulation to modify cerebral inflammation. The hypothalamic-pituitary-thyroid (HPT) axis was inhibited in <i>L</i>. <i>crocea</i> brains during the early period of hypoxia, thus leading to a decrease in thyroid hormone (T3 and T4) production. Decrease of HPT axis-thyroid hormones subsequently may inhibit protein synthesis by the PI3K-Akt-mTOR-S6K signaling pathway, which contributes to the reduction in energy consumption during hypoxia stress. Meanwhile, down-regulation of HPT axis-thyroid hormones also represses the tricarboxylic acid (TCA) cycle and accelerates the anaerobic glycolytic pathway, which facilitates O₂-independent ATP production under hypoxia. Therefore the HPT axis-mediated effects may play roles in response to hypoxia by reorganizing energy consumption and generation. Genes related to the neuro-endocrine system (orange), immunity (red), and metabolic system and protein synthesis (blue) are indicated. The outer border indicates the brain of <i>L</i>. <i>crocea</i>. The arrow represents promotion, and the interrupted line represents inhibition. Solid lines indicate direct relationships between genes. Dashed lines indicate that more than one step is involved in the process.</p

Characterisation of the T-cell lineages in <i>L</i>. <i>crocea</i> adaptive immunity and the expanded genes in antiviral immunity.

<p>(<b>A</b>) A schematic diagram summarising genes related to different T-cell lineages in <i>L</i>. <i>crocea</i> is shown. The inducible factors, the main regulatory transcriptional factors, and the immune effectors of T cells are present in green, blue, and orange backgrounds, respectively. The genes that have been annotated by genome survey are shown in black, and the unannotated genes are shown in red. IL-2R represents all three subunit genes of <i>L</i>. <i>crocea</i> IL-2 receptor, IL-2RB, IL-2RG and IL-2RA/ IL-15RA genes. The majority of the CD4⁺ T-helper type 1 (Th1), CD4⁺ T-helper type 2 (Th2) and CD8⁺ T cell-related genes have been found in the <i>L</i>. <i>crocea</i> genome. The genes related to Th17 cell- and γδ-T cell-mediated mucosal immunity are also conserved in <i>L</i>. <i>crocea</i>. (<b>B</b>) Several key genes are expanded in the antiviral immunity pathways in <i>L</i>. <i>crocea</i>. The genes that have been identified in the <i>L</i>. <i>crocea</i> genome are shown in orange boxes, and the lost gene (<i>RIG-I</i>) is shown in the grey box. LGP2 is able to bind to double-stranded RNA (dsRNA) to trigger interferon production, but the adaptor molecule of LGP2 is still unknown in fish. The red boxes indicate gene families (<i>TRIM25</i>, <i>cGAS</i>, <i>DDX41</i>, and <i>NLRC3</i>) that are expanded in <i>L</i>. <i>crocea</i>. The arrow represents induction, and the interrupted line represents inhibition.</p

Summary of the <i>Larimichthys crocea</i> genome

<p>BAC = bacterial artificial chromosome; WGS = whole genome shotgun.</p><p>Summary of the <i>Larimichthys crocea</i> genome</p

Phylogenetic tree of and orthologous genes in <i>L</i>. <i>crocea</i> and other vertebrates.

<p>(<b>A</b>) The phylogenetic tree was constructed from 2,257 single-copy genes with 3.18 M reliable sites by maximum likelihood methods. The red points on six of the internal nodes indicate fossil calibration times in the analysis. Blue numbers indicate the divergence time (Myr, million years ago), and the green and red numbers represent the expanded and extracted gene families, respectively, in <i>L</i>. <i>crocea</i>. (<b>B</b>) The different types of orthologous relationships are shown. “1:1:1” = universal single-copy genes; “N:N:N” = orthologues exist in all genomes; “Fish” = fish-specific genes; “SD” = genes that have undergone species-specific duplication; “Homology” = genes with an e-value less than 1e-5 by BLAST but do not cluster to a gene family; “ND” = species-specific genes; and “Others” = orthologues that do not fit into the other categories. (<b>C</b>) The shared and unique gene families in five teleost fish are shown in the Venn diagram. (<b>D</b>) Distribution of the identity values of orthologous genes is compared among <i>L</i>. <i>crocea</i> and other teleosts.</p