Yicheng Zhao

Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system

Here, we report a convenient and efficient miRNA inhibition strategy employing the CRISPR system. Using specifically designed gRNAs, miRNA gene has been cut at a single site by Cas9, resulting in knockdown of the miRNA in murine cells. Using a modified CRISPR interference system (CRISPRi), inactive Cas9 can reversibly prevent the expression of both monocistronic miRNAs and polycistronic miRNA clusters. Furthermore, CRISPR/CRISPRi is also capable of suppressing genes in porcine cells.

Circulating microRNAs: Promising Biomarkers Involved in Several Cancers and Other Diseases

Recently, many studies indicated that microRNAs (miRNAs) stably existed in various body fluids, including serum, plasma, saliva, and urine. Such miRNAs that exist in mammalian body fluids are known as circulating miRNAs, and they can transmit signals between cells and regulate intracellular gene expression. Currently, we barely understand the characteristics, sources, secretion, uptake, and functions of newly generated miRNAs. Particularly, it has been shown that certain types of circulating miRNAs can provide effective clinical data, suggesting their roles as novel biomarkers for the early detection of diseases such as cancers, cardiovascular diseases, and diabetes. Therefore, miRNAs have attracted much attention in academia for their promising applications in fundamental research and clinical diagnosis. This review summarizes some of the functional studies that have been conducted as well as the promising applications of circulating miRNAs, and we hope it will benefit other researchers in this field.

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.

Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways *#

RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous micro-RNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.

Inhibiting cyprinid herpesvirus-3 replication with CRISPR/Cas9

ObjectivesThe potential of CRISPR/Cas9 gene editing to repress CyHV-3 was tested in vitro.ResultsBy targeting two basic target genes necessary for the early transcription of CyHV-3, we show that virus transcription and particle release were significantly decreased by CRISPR/Cas9, as measured by quantitative real-time PCR and virus titration experiments, respectively.Conclusions(A) The effectiveness is confirmed of the CRISPR/Cas9 system at repressing exogenous genes, including large viral genomic DNA, by introducing site-specific mutations in vitro. (B) The CyHV-3 virus replicates poorly in Cas9-positive cells

miR-18a counteracts AKT and ERK activation to inhibit the proliferation of pancreatic progenitor cells

Activation of endogenous stem/progenitor cells to repair injured tissues is an ideal option for disease treatment. However, adult pancreatic progenitor cells remain in a quiescent state in vivo. Thus, it is difficult to stimulate proliferation and differentiation in these progenitor cells, and the cause remains elusive. miR-17-92 cluster miRNAs are highly conserved in mammals and are expressed in multiple tissue stem/progenitor cells, but their role in pancreatic progenitor cells are less well known. In the present study, we demonstrate that miR-18a, but not the other members of the miR-17-92 gene cluster, inhibits the proliferation of pancreatic progenitor cells in vitro and ex vivo. miR-18a inhibits proliferation of adult pancreatic progenitor cells through arresting the cell cycle at G1 stage, indicating that miR-18a plays a role in keeping the adult pancreatic progenitor cells in quiescence. miR-18a inhibits pancreatic progenitor proliferation by targeting the gene expressions of connective tissue growth factor (CTGF), neural precursor cell expressed, developmentally down-regulated 9 (Nedd9), and cyclin dependent kinase 19 (CDK19), as well as by suppressing activation of the proliferation-related signaling pathways phosphatidylinositol 3-kinase–protein kinase B (PI3K/AKT) and extracellular signal-regulated kinase (ERK).

Classical swine fever virus replicated poorly in cells from MxA transgenic pigs

BackgroundIn addition to their value as livestock, pigs are susceptible to classical swine fever virus (CSFV) and can serve as reservoirs for CSFV, allowing it to develop into an epizootic. CSFV, a pestivirus of the Flaviviridae family, has a single-stranded RNA genome. Recent research has indicated that the human MxA protein inhibits the life cycles of certain RNA viruses, such as members of the Bunyaviridae family, the Flaviviridae family and others.ResultsTo produce pigs with antiviral protection against CSFV, transgenic pigs expressing human MxA were generated by nuclear transplantation. Cells from three MxA transgenic piglets were used to investigate in vitro antiviral activity of MxA aganist CSFV, and the results of in vitro indirect immunofluorescence assays, virus titration and real-time PCR indicated that the MxA transgenic pig has an antiviral capacity against CSFV.ConclusionsTransgene with human MxA on pigs is feasible. High levels of MxA expression do inhibit CSFV in vitro at early time points post-infection at 60-96dpi.

Large-scale prediction of ADAR-mediated effective human A-to-I RNA editing

Abstract Adenosine‐to‐inosine (A‐to‐I) editing by adenosine deaminase acting on the RNA (ADAR) proteins is one of the most frequent modifications during post‐ and co‐transcription. To facilitate the assignment of biological functions to specific editing sites, we designed an automatic online platform to annotate A‐to‐I RNA editing sites in pre‐mRNA splicing signals, microRNAs (miRNAs) and miRNA target untranslated regions (3′ UTRs) from human (Homo sapiens) high‐throughput sequencing data and predict their effects based on large‐scale bioinformatic analysis. After analysing plenty of previously reported RNA editing events and human normal tissues RNA high‐seq data, >60 000 potentially effective RNA editing events on functional genes were found. The RNA Editing Plus platform is available for free at https://www.rnaeditplus.org/, and we believe our platform governing multiple optimized methods will improve further studies of A‐to‐I‐induced editing post‐transcriptional regulation.