Biology subjects

Zhang, F.

Publications and source records attributed to Zhang, F..

467 records · Page 26Linked to original sources

bioRxiv2017

Causal associations between risk factors and common diseases inferred from GWAS summary data

Health risk factors such as body mass index (BMI), serum cholesterol and blood pressure are associated with many common diseases. It often remains unclear whether the risk factors are cause or consequence of disease, or whether the associations are the result of confounding. Genetic methods are useful to infer causality because genetic variants are present from birth and therefore unlikely to be confounded with environmental factors. We develop and apply a method (GSMR) that performs a multi-SNP Mendelian Randomization analysis using summary-level data from large genome-wide association studies (sample sizes of up to 405,072) to test the causal associations of BMI, waist-to-hip ratio, serum cholesterols, blood pressures, height and years of schooling (EduYears) with a range of common diseases. We identify a number of causal associations including a protective effect of LDL-cholesterol against type-2 diabetes (T2D) that might explain the side effects of statins on T2D, a protective effect of EduYears against Alzheimers disease, and bidirectional associations with opposite effects (e.g. higher BMI increases the risk of T2D but the effect T2D of BMI is negative). HDL-cholesterol has a significant risk effect on age-related macular degeneration, and the effect size remains significant accounting for the other risk factors. Our study develops powerful tools to integrate summary data from large studies to infer causality, and provides important candidates to be prioritized for further studies in medical research and for drug discovery.

genetics↗

bioRxiv2017

GUIDES: sgRNA design for loss-of-function screens

GUIDES (Graphical User Interface for DNA Editing Screens) is a web-based tool for the design of custom, large-scale CRISPR libraries for loss-of-function screens in human and mouse. GUIDES combines multi-tissue RNA-sequencing data to target expressed exons, protein annotation to target functional domains, sophisticated on-target and off-target guide RNA scoring and other optimizations to create CRISPR libraries directly from a list of genes without requiring any programming expertise.

genetics↗

bioRxiv2017

CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells

The activation or repression of a genes expression is primarily controlled by changes in the proteins that occupy its regulatory elements. The most common method to identify proteins associated with genomic loci is chromatin immunoprecipitation (ChIP). While having greatly advanced our understanding of gene expression regulation, ChIP requires specific, high quality, IP-competent antibodies against nominated proteins, which can limit its utility and scope for discovery. Thus, a method able to discover and identify proteins associated with a particular genomic locus within the native cellular context would be extremely valuable. Here, we present a novel technology combining recent advances in chemical biology, genome targeting, and quantitative mass spectrometry to develop genomic locus proteomics, a method able to identify proteins which occupy a specific genomic locus.

biochemistry↗

bioRxiv2017

Direct in vivo mapping of functional suppressors in glioblastoma genome

Glioblastoma (GBM) is one of the deadliest cancers, with limited effective treatments and single-digit five-year survival 1-7. A causative understanding of genetic factors that regulate GBM formation is of central importance 8-19. However, a global, quantitative and functional understanding of gliomagenesis in the native brain environment has been lacking due to multiple challenges. Here, we developed an adeno-associated virus (AAV) mediated autochthonous CRISPR screen and directly mapped functional suppressors in the GBM genome. Stereotaxic delivery of an AAV library targeting significantly mutated genes into fully immunocompetent conditional Cas9 mice robustly led to gliomagenesis, resulting in tumors that recapitulate features of human GBM. Targeted capture sequencing revealed deep mutational profiles with diverse patterns across mice, uncovering in vivo roles of previously uncharacterized factors in GBM such as immune regulator B2m, zinc finger protein Zc3h13, transcription repressor Cic, epigenetic regulators Mll2/3 and Arid1b, alongside canonical tumor suppressors Nf1 and Pten. Comparative cancer genomics showed that the mutation frequencies across all genes tested in mice significantly correlate with those in human from two independent patient cohorts. Co-mutation analysis identified frequently co-occurring driver combinations, which were validated using AAV minipools, such as Mll2, B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1. Distinct from Nf1-oncotype tumors, Rb1-oncotype tumors exhibit undifferentiated histopathology phenotype and aberrant activation of developmental reprogramming signatures such as Homeobox gene clusters. The secondary addition of Zc3h13 or Pten mutations drastically altered the gene expression profiles of Rb1 mutants and rendered them more resistant to the GBM chemotherapeutic temozolomide. Our study provides a systematic functional landscape of GBM suppressors directly in vivo, opening new paths for high-throughput molecular mapping and cancer phenotyping.

systems biology↗

bioRxiv2017

Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing

High-resolution, high-throughput direct in vivo screening of functional genetic factors in native tissues has long been challenging. Adeno-associated viruses (AAV) are powerful carriers of transgenes and have been shown to mediate efficient genome editing in various organs in mice. Here, we developed a new technological approach, Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing (PASTAS), and demonstrated its application for directly mapping functional cancer driver variants in the mouse liver in an autochthonous manner. Intravenous delivery of an AAV-CRISPR library targeting a set of the most frequently mutated tumor suppressor genes into fully immunocompetent conditional Cas9 knock-in mice consistently generated highly complex autochthonous liver tumors. The molecular landscapes of these genetically diverse tumors were mapped out by deep direct readout of Cas9-generated variants at predicted sgRNA cut sites using molecular inversion probe sequencing. Co-occurrence and correlation analyses as well as validation with lower complexity minipools further confirmed the potency of various co-mutated drivers. The PASTAS method can be applied to virtually any gene sets, any cancer types, or any type of in vivo genetic studies other than cancer.

systems biology↗

bioRxiv2017

Karyotype stability and unbiased fractionation in the paleo-allotetraploid Cucurbita genomes

The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Interspecific hybrids between C. maxima and C. moschata are widely used as rootstocks for other cucurbit crops. We report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetraploidization event in Cucurbita. We are able to partition the genome into two homoeologous subgenomes based on different genetic distances to melon, cucumber and watermelon in the Benincaseae tribe. We estimate that the two diploid progenitors successively diverged from Benincaseae around 31 and 26 million years ago (Mya), and the allotetraploidization happened earlier than 3 Mya, when C. maxima and C. moschata diverged. The subgenomes have largely maintained the chromosome structures of their diploid progenitors. Such long-term karyotype stability after polyploidization is uncommon in plant polyploids. The two subgenomes have retained similar numbers of genes, and neither subgenome is globally dominant in gene expression. Allele-specific expression analysis in the C. maxima x C. moschata interspecific F1 hybrid and the two parents indicates the predominance of trans-regulatory effects underlying expression divergence of the parents, and detects transgressive gene expression changes in the hybrid correlated with heterosis in important agronomic traits. Our study provides insights into plant genome evolution and valuable resources for genetic improvement of cucurbit crops.

genomics↗

bioRxiv2017

A Survey of Genome Editing Activity for 16 Cpf1 orthologs

The recently discovered class 2 CRISPR-Cas endonuclease Cpf1 offers several advantages over Cas9, including the ability to process its own array and requirement for just a single RNA guide. These attributes make Cpf1 promising for many genome engineering applications. To further expand the suite of Cpf1 tools available, we tested 16 Cpf1 orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cpf1), exhibited robust indel formation. We also show that Mb3Cpf1 displays some tolerance for a shortened PAM (TTN versus the canonical Cpf1 PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.

molecular biology↗

bioRxiv2017

Integrated Computational Guide Design, Execution, And Analysis Of Arrayed And Pooled CRISPR Genome Editing Experiments

CRISPR 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 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, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. 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 in 1-2 days that can be performed by both computational and non-computational biologists alike.

molecular biology↗

bioRxiv2017

Early urinary candidate biomarker discovery in a rat thioacetamide-induced liver fibrosis model

Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis, which results from the deposition of extracellular matrix (ECM) components, is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide (TAA) liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS). A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins in the TAA 1-week, 3-week, 6-week and 8-week groups. A total of 40 urinary proteins were statistically significant (fold change >2 and p<0.05), 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase (ALT) and aspartate transaminase (AST) changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Massons staining.

molecular biology↗

bioRxiv2017

Early Candidate Biomarkers Found From Urine Of Astrocytoma Rat Before Changes In MRI

Astrocytoma is the most common aggressive glioma and its early diagnosis remains difficult. Biomarkers are changes associated with the disease. Urine, which is not regulated by homeostatic mechanisms, accumulates changes and therefore is a better source for biomarker discovery. In this study, C6 cells were injected into Wistar rats brain as astrocytoma model. Urine samples were collected at day 2, day 6, day 10 and day 13 after injection, and the urinary proteomes were analyzed. On the 10th day, lesions appeared in magnetic resonance imaging. On the 13th day, clinical symptoms started. But differential urinary proteins were changed with the development of the astrocytoma, and can provide clues even on the 2nd and 6th day. Twenty-seven differential proteins with human orthologs had been reported to associate with astrocytoma. Thirty-nine proteins were verified in four more rats as candidate biomarkers of astrocytoma using multiple-reaction monitoring. A panel of differential urinary proteins may provide early biomarkers for diagnose of astrocytoma.

biochemistry↗

bioRxiv2017

DroNc-Seq: Deciphering cell types in human archived brain tissues by massively-parallel single nucleus RNA-seq

Single nucleus RNA-Seq (sNuc-Seq) profiles RNA from tissues that are preserved or cannot be dissociated, but does not provide the throughput required to analyse many cells from complex tissues. Here, we develop DroNc-Seq, massively parallel sNuc-Seq with droplet technology. We profile 29,543 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient and unbiased classification of cell types, paving the way for charting systematic cell atlases.

genomics↗

bioRxiv2017

A Multi-Parameter Analysis of Cellular Coordination of Multiple Transcriptome Regulation Mechanisms

To understand cellular coordination of multiple transcriptome regulation mechanisms, we simultaneously measured transcription rate (TR), mRNA abundance (RA) and translation activity (TA). This revealed multiple quantitative insights. First, the genomic profiles of the three parameters are systematically different in key statistical features. Sequentially more genes exhibit extreme low or high expression values from TR to RA, then to TA. That is, because of cellular coordination of these regulatory mechanisms, sequentially higher levels of gene expression selectivity are achieved as genetic information flow from the genome to the proteome. Second, the contribution of the stabilization-by-translation regulatory mechanism to the cellular coordination process was assessed. The data enabled an estimation of mRNA stability, revealing a moderate but significant positive correlation between the estimated mRNA stability and translation activity. Third, the proportion of a mRNA occupied by un-translated regions (UTR) exhibits a negative relationship with the level of this correlation, and is thus a major determinant of the mode of regulation of the mRNA. High-UTR-proportion mRNAs tend to defy the stabilization-by-translation regulatory mechanism, staying out of the polysome but remaining stable; mRNAs with little UTRs largely follow this regulation. In summary, we quantitatively delineated the relationship among multiple transcriptome regulation parameters, i.e., cellular coordination of corresponding regulatory mechanisms.

systems biology↗

bioRxiv2017

HiCRep: assessing the reproducibility of Hi-C data using a stratum-adjusted correlation coefficient

Hi-C is a powerful technology for studying genome-wide chromatin interactions. However, current methods for assessing Hi-C data reproducibility can produce misleading results because they ignore spatial features in Hi-C data, such as domain structure and distance dependence. We present HiCRep, a framework for assessing the reproducibility of Hi-C data that systematically accounts for these features. In particular, we introduce a novel similarity measure, the stratum adjusted correlation coefficient (SCC), for quantifying the similarity between Hi-C interaction matrices. Not only does it provide a statistically sound and reliable evaluation of reproducibility, SCC can also be used to quantify differences between Hi-C contact matrices and to determine the optimal sequencing depth for a desired resolution. The measure consistently shows higher accuracy than existing approaches in distinguishing subtle differences in reproducibility and depicting interrelationships of cell lineages. The proposed measure is straightforward to interpret and easy to compute, making it well-suited for providing standardized, interpretable, automatable, and scalable quality control. The freely available R package HiCRep implements our approach.

bioinformatics↗

bioRxiv2017

Stability and function of a putative microtubule organizing center in the human parasite Toxoplasma gondii

The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here, we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and discovered that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 ({Delta}kinesinA{Delta}apr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live{Delta} kinesinA{Delta}apr1 parasites, and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and to invade host cells, as well as decreased secretion of effectors important for these processes. Together, the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

cell biology↗

bioRxiv2016

Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

microbiology↗

bioRxiv2016

Engineered Cpf1 Enzymes with Altered PAM Specificities

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

synthetic biology↗

bioRxiv2016

BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ

We present a method for genome-wide DNA double-strand Breaks (DSBs) Labeling In Situ and Sequencing (BLISS) which, compared to existing methods, introduces several key features: 1) high efficiency and low input requirement by in situ DSB labeling in cells or tissue sections directly on a solid surface; 2) easy scalability by performing in situ reactions in multi-well plates; 3) high sensitivity by linearly amplifying tagged DSBs using in vitro transcription; and 4) accurate DSB quantification and control of PCR biases by using unique molecular identifiers. We demonstrate the ability to use BLISS to quantify natural and drug-induced DSBs in low-input samples of cancer cells, primary mouse embryonic stem cells, and mouse liver tissue sections. Finally, we applied BLISS to compare the specificity of CRISPR-associated RNA-guided endonucleases Cas9 and Cpf1, and found that Cpf1 has higher specificity than Cas9. These results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

molecular biology↗