Showing papers by "Feng Zhang published in 2018"

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

Abstract: Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

Abstract: Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.

Field-deployable viral diagnostics using CRISPR-Cas13

Abstract: Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in

Engineered CRISPR-Cas9 nuclease with expanded targeting space

Abstract: The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non–base-specific interactions, thereby enabling the NG PAM recognition We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells

RNA editing with CRISPR-Cas13

Abstract: Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products.TypeVI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13.We profiled typeVI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells.This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations.We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.

FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in obesity.

Abstract: Background Obesity-induced chronic inflammation is critical in the pathogenesis of insulin resistance, and the recruitment and proinflammatory activation of adipose tissue macrophages (ATMs) is important for the development of this process. Here, we examined the effects of fibronectin type III domain-containing 5 (FNDC5) on inflammation and insulin resistance in high-fat diet-induced obese mice. Materials and Methods Male wild-type (WT) and FNDC5−/− mice were fed with standard chow (Ctrl) or high fat diet (HFD) for 20 weeks to induce obesity and insulin resistance. Firstly, effects of FNDC5 gene deletion on obesity, insulin resistance, macrophage accumulation and polarization and adipose tissue inflammation were determined in mice. Secondly, the macrophage polarity shift was further examined with flow cytometry in isolated stromal vascular fraction (SVF). Thirdly, the effects of exogenous FNDC5 on lipopolysaccharide (LPS)-induced macrophage polarization, inflammation and the underlying signaling mechanism were investigated in RAW264.7 macrophages and primary mouse peritoneal cavity macrophages (PMs). Finally, the therapeutic effects of FNDC5 overexpression were examined in HFD-induced obese WT and FNDC5−/− mice. Results FNDC5 gene deletion aggravated obesity, insulin resistance, fat accumulation and inflammation accompanied with enhanced AMPK inhibition, macrophages recruitment and M1 polarization in mice fed with HFD. Exogenous FNDC5 inhibited LPS-induced M1 macrophage polarization and inflammatory cytokine production via AMPK phosphorylation in both RAW264.7 macrophages and PMs. FNDC5 overexpression attenuated insulin resistance, AMPK inhibition, M1 macrophage polarization and inflammatory cytokine production in adipose tissue of obese WT and FNDC5−/− mice. Conclusions FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in HFD-induced obesity. FNDC5 plays several beneficial roles in obesity and may be used as a therapeutic regimen for preventing inflammation and insulin resistance in obesity and diabetes.

The nuclear matrix protein HNRNPU maintains 3D genome architecture globally in mouse hepatocytes.

Abstract: Eukaryotic chromosomes are folded into higher-order conformations to coordinate genome functions. In addition to long-range chromatin loops, recent chromosome conformation capture (3C)-based studies have indicated higher levels of chromatin structures including compartments and topologically associating domains (TADs), which may serve as units of genome organization and functions. However, the molecular machinery underlying these hierarchically three-dimensional (3D) chromatin architectures remains poorly understood. Via high-throughput assays, including in situ Hi-C, DamID, ChIP-seq, and RNA-seq, we investigated roles of the Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), a nuclear matrix (NM)-associated protein, in 3D genome organization. Upon the depletion of HNRNPU in mouse hepatocytes, the coverage of lamina-associated domains (LADs) in the genome increases from 53.1% to 68.6%, and a global condensation of chromatin was observed. Furthermore, disruption of HNRNPU leads to compartment switching on 7.5% of the genome, decreases TAD boundary strengths at borders between A (active) and B (inactive) compartments, and reduces chromatin loop intensities. Long-range chromatin interactions between and within compartments or TADs are also significantly remodeled upon HNRNPU depletion. Intriguingly, HNRNPU mainly associates with active chromatin, and 80% of HNRNPU peaks coincide with the binding of CTCF or RAD21. Collectively, we demonstrated that HNRNPU functions as a major factor maintaining 3D chromatin architecture, suggesting important roles of NM-associated proteins in genome organization.

The power of synthetic biology for bioproduction, remediation and pollution control: The UN's Sustainable Development Goals will inevitably require the application of molecular biology and biotechnology on a global scale

Abstract: EMBO Reports (2018) e45658 The agenda of the UN's Sustainable Development Goals (SDGs) [1] challenges the synthetic biology community—and the life sciences as a whole—to develop transformative technologies that help to protect, even expand our planet's habitability. While modern tools for genome editing already benefit applications in health and agriculture, sustainability also asks for a dramatic transformation of our use of natural resources. The challenge is not just to limit and, wherever possible revert emissions of pollutants and greenhouse gases, but also to replace environmentally costly processes based on fossil fuels with bio‐based sustainable alternatives. This task is not exclusively a scientific and technical one but will also require guidelines and regulations for the development and large‐scale deployment of this new type of bio‐based production. Some recent advances that can (or soon could) enable us to make progress in these areas—and several possible governance principles—need to be addressed. ### The potential of biotechnology The transformative power of modern, science‐based biotechnology that started in the late 1970s has been accelerated by recent developments, such as massive DNA synthesis/sequencing, systems and synthetic biology, and CRISPR tools for genome editing. The interface of these disciplines and techniques with other flagship technologies of the ongoing Fourth Industrial Revolution [2], such as artificial intelligence, robotics, big data, ITs, and so on, will usher in a society, economy and industry that are very different from what we know today. So far, market forces have pushed most research efforts towards health‐related issues and agricultural productivity, as these areas can more easily harvest low‐hanging fruits of contemporary systems‐based biotechnology. But the spectacular advances in biomedicine and agricultural technologies are happening during an acute global environmental crisis caused by overpopulation, loss of biodiversity, greenhouse gas emissions and pollution. Thus, environmental sustainability …

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

Abstract: CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR–mediated direct in vivo screening

Abstract: Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adeno-associated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and died within 4 months. We used molecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo.

The coexistence of copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) at a locus can result in distorted calculations of the significance in associating SNPs to disease.

Abstract: With the recent advance in genome-wide association studies (GWAS), disease-associated single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) have been extensively reported. Accordingly, the issue of incorrect identification of recombination events that can induce the distortion of multi-allelic or hemizygous variants has received more attention. However, the potential distorted calculation bias or significance of a detected association in a GWAS due to the coexistence of CNVs and SNPs in the same genomic region may remain under-recognized. Here we performed the association study within a congenital scoliosis (CS) cohort whose genetic etiology was recently elucidated as a compound inheritance model, including mostly one rare variant deletion CNV null allele and one common variant non-coding hypomorphic haplotype of the TBX6 gene. We demonstrated that the existence of a deletion in TBX6 led to an overestimation of the contribution of the SNPs on the hypomorphic allele. Furthermore, we generalized a model to explain the calculation bias, or distorted significance calculation for an association study, that can be 'induced' by CNVs at a locus. Meanwhile, overlapping between the disease-associated SNPs from published GWAS and common CNVs (overlap 10%) and pathogenic/likely pathogenic CNVs (overlap 99.69%) was significantly higher than the random distribution (p < 1 × 10-6 and p = 0.034, respectively), indicating that such co-existence of CNV and SNV alleles might generally influence data interpretation and potential outcomes of a GWAS. We also verified and assessed the influence of colocalizing CNVs to the detection sensitivity of disease-associated SNP variant alleles in another adolescent idiopathic scoliosis (AIS) genome-wide association study. We proposed that detecting co-existent CNVs when evaluating the association signals between SNPs and disease traits could improve genetic model analyses and better integrate GWAS with robust Mendelian principles.

Crispr effector system based diagnostics

Abstract: The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect broth DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of disease-associated cell free DNA.

Conferring DNA virus resistance with high specificity in plants using virus-inducible genome-editing system

Abstract: The CRISPR/Cas9 system has recently been engineered to confer resistance to geminiviruses in plants. However, we show here that the usefulness of this antiviral strategy is undermined by off-target effects identified by deep sequencing in Arabidopsis. We construct two virus-inducible CRISPR/Cas9 vectors that efficiently inhibit beet severe curly top virus (BSCTV) accumulation in both transient assays (Nicotiana benthamiana) and transgenic lines (Arabidopsis). Deep sequencing detects no off-target effect in candidate sites of the transgenic Arabidopsis. This kind of virus-inducible genome-editing system should be widely applicable for generating virus-resistant plants without off-target costs.

Endoplasmic reticulum resident oxidase ERO1-Lalpha promotes hepatocellular carcinoma metastasis and angiogenesis through the S1PR1/STAT3/VEGF-A pathway

Abstract: Mounting evidence demonstrates that expression of ERO1α, an endoplasmic reticulum (ER)-resident oxidase, is a poor prognosis factor in a variety of human cancers. However, the clinical relevance of ERO1α and its molecular mechanisms underlying tumor progression have not been determined for hepatocellular carcinoma (HCC). ERO1α expression levels in HCC tissues and cells were detected by quantitative real-time PCR and western blotting. ERO1α shRNAs and overexpression vector were transfected into HCC cells to downregulate or upregulate ERO1α expression. In vitro and in vivo assays were performed to investigate the function of ERO1α in invasion, metastasis, and angiogenesis of HCC. We found high ERO1α expression in HCC tissues and cells that was significantly associated with metastasis and poor clinicopathologic features of vascular invasion, advanced Edmondson Grade, and TNM stage. Loss-of-function and gain-of-function studies showed that ERO1α prompted migration, invasion, epithelial–mesenchymal transition (EMT), and angiogenesis of HCC cells both in vitro and in vivo. Further studies verified a positive correlation between ERO1α and S1PR1, upregulated in metastatic HCC tissues compared with HCC tissues without metastasis. S1PR1 knockdown markedly diminished the effects of ERO1α on HCC cell migration, invasion and vascular endothelial growth factor (VEGF) expression. Most importantly, ERO1α knockdown significantly repressed the death of HCC xenograft mouse models by reducing tumor distant metastasis, and host angiogenesis by suppressing the expression of S1PR1, p-STAT3, and VEGF-A in HCC cells. Our findings suggest that ERO1α is significantly correlated with reduced survival and poor prognosis, and promotes HCC metastasis and angiogenesis by triggering the S1PR1/STAT3/VEGF-A signaling pathway. ERO1α might be a novel candidate in HCC prognosis and therapy.

Ore genesis and geodynamic setting of the Lianhuashan porphyry tungsten deposit, eastern Guangdong Province, SE China: constraints from muscovite 40 Ar- 39 Ar and zircon U-Pb dating and Hf isotopes

Abstract: The Lianhuashan deposit has long been regarded as a typical tungsten porphyry deposit, located in the eastern Guangdong Province, in the Southeastern Coastal Metallogenic Belt (SCMB). LA–MC–ICP–MS zircon U–Pb dating of the quartz porphyry yielded a weighted mean 206Pb/238U age of 137.3 ± 2.0 Ma, which is interpreted as the emplacement age of the quartz porphyry. Hydrothermal muscovite yielded a plateau 40Ar/39Ar age of 133.2 ± 0.9 Ma, which is consistent with the zircon U–Pb age, suggesting that the tungsten mineralization is genetically related to the quartz porphyry. Combined with previous studies, we suggest that there is a 145–135 Ma episode linking the granitic magmas with W–Sn ore systems in the SCMB. Zircon eHf (t) values of the quartz porphyry are in range of − 3.8 to 0.9, and the two-stage Hf model ages (TDM2) are 1.1–1.4 Ga, which is younger than the basement rocks in the Cathaysia Block (1.8–2.2 Ga), signifying that the quartz porphyry was predominantly derived from melting of Mesoproterozoic crust containing variable amounts of mantle components. In combination with the newly recognized coeval alkaline/bimodal magmatism and A-type granites in eastern Guangdong, we suggest that the 145–135 Ma W–Sn metallogenic event of the SCMB is related to a geodynamic setting of large-scale lithospheric extension and thinning, which can be ascribed to melting of the crust caused by mantle upwelling, triggered by the oblique subduction of the Izanagi plate.

Systems, methods, and compositions for targeted nucleic acid editing

Abstract: The disclosure provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Casl3 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.

DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

Abstract: Analyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in profiling the molecular constituents of cells, spatially mapping these constituents remains a disjointed and machinery-intensive process, relying on either light microscopy or direct physical registration and capture. Here, we demonstrate DNA microscopy, a new imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences, and infers physical images of the original transcripts at cellular resolution. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.

A Genome-wide Expression Association Analysis Identifies Genes and Pathways Associated with Amyotrophic Lateral Sclerosis.

Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with strong genetic components. To identity novel risk variants for ALS, utilizing the latest genome-wide association studies (GWAS) and eQTL study data, we conducted a genome-wide expression association analysis by summary data-based Mendelian randomization (SMR) method. Summary data were derived from a large-scale GWAS of ALS, involving 12577 cases and 23475 controls. The eQTL annotation dataset included 923,021 cis-eQTL for 14,329 genes and 4732 trans-eQTL for 2612 genes. Genome-wide single gene expression association analysis was conducted by SMR software. To identify ALS-associated biological pathways, the SMR analysis results were further subjected to gene set enrichment analysis (GSEA). SMR single gene analysis identified one significant and four suggestive genes associated with ALS, including C9ORF72 (P value = 7.08 × 10−6), NT5C3L (P value = 1.33 × 10−5), GGNBP2 (P value = 1.81 × 10−5), ZNHIT3(P value = 2.94 × 10−5), and KIAA1600(P value = 9.97 × 10−5). GSEA identified 7 significant biological pathways, such as PEROXISOME (empirical P value = 0.006), GLYCOLYSIS_GLUCONEOGENESIS (empirical P value = 0.043), and ARACHIDONIC_ACID_ METABOLISM (empirical P value = 0.040). Our study provides novel clues for the genetic mechanism studies of ALS.

SKP2 Promotes Hepatocellular Carcinoma Progression Through Nuclear AMPK-SKP2-CARM1 Signaling Transcriptionally Regulating Nutrient-Deprived Autophagy Induction.

Abstract: Background/aims SKP2 overexpression has been associated with poor prognosis in numerous cancers The mechanisms of autophagy in the tumor pathogenesis have been a research focus recently How the SKP2 involved in autophagy expresses oncogenic characteristics, especially in HCC, are largely unclear Methods The expression of SKP2 was detected by qPCR, Western blot, Immunohistochemical (IHC) and Immunofluorescence (IF) techniques SKP2 was knocked down or overexpressed by lentivirus transfection in HCC cells Functional assays such as CCK8 assays, transwell migration and invasion assays, and colony formation assays were performed to determine the role of SKP2 in HCC Furthermore, autophagy was induced by glucose deprivation in HCC cells followed by monitoring of the levels and distributions of SKP2, CARM1 and AMPK Results Our data showed that SKP2 levels were significantly increased in HCC cell lines and HCC tissues rather than corresponding normal liver tissues, and augmented SKP2 levels were statistically correlated with tumor grade, size and metastases By up-regulation or down-regulation of SKP2 in HCC cells, we confirmed that SKP2 encourages proliferation, migration, invasion, and colony formation We then found that SKP2 was inhibited, CARM1 increased and AMPKα2 became activated in the nucleus under glucose deprivation induced autophagy Moreover, we discovered that SKP2 was repressing CARM1 in the nucleus under nutrient-sufficient conditions in HCC Conclusions We show that SKP2 promotes HCC progression and its nuclear functions of autophagy induction with CARM1 and AMPK, which may provide a potential target for HCC therapy

Crispr/cas-cytidine deaminase based compositions, systems, and methods for targeted nucleic acid editing

Abstract: The invention provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.

Novel type vi crispr orthologs and systems

Abstract: The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the disclosure provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.