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RSS Most recent PubMed article (full bibliography available above)

  • Using CRISPR to understand and manipulate gene regulation April 29, 2021
    Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic […]
    Ersin Akinci
  • Detection of gene cis-regulatory element perturbations in single-cell transcriptomes March 12, 2021
    We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss […]
    Grace Hui Ting Yeo
  • Machine learning based CRISPR gRNA design for therapeutic exon skipping January 8, 2021
    Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent […]
    Wilson Louie
  • Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes December 30, 2020
    Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing-issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a […]
    Vedagopuram Sreekanth
  • Comprehensive Mapping of Key Regulatory Networks that Drive Oncogene Expression November 25, 2020
    Gene expression is controlled by the collective binding of transcription factors to cis-regulatory regions. Deciphering gene-centered regulatory networks is vital to understanding and controlling gene misexpression in human disease; however, systematic approaches to uncovering regulatory networks have been lacking. Here we present high-throughput interrogation of gene-centered activation networks (HIGAN), a pipeline that employs a suite […]
    Lin Lin
  • Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay September 25, 2020
    A key mechanism in cellular regulation is the ability of the transcriptional machinery to physically access DNA. Transcription factors interact with DNA to alter the accessibility of chromatin, which enables changes to gene expression during development or disease or as a response to environmental stimuli. However, the regulation of DNA accessibility via the recruitment of […]
    Jennifer Hammelman
  • A High-Throughput Genome-Integrated Assay Reveals Spatial Dependencies Governing Tcf7l2 Binding September 10, 2020
    Predicting where transcription factors bind in the genome from their in vitro DNA-binding affinity is confounded by the large number of possible interactions with nearby transcription factors. To characterize the in vivo binding logic for the Wnt effector Tcf7l2, we developed a high-throughput screening platform in which thousands of synthesized DNA phrases are inserted into […]
    Tomasz Szczesnik
  • Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics September 2, 2020
    The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness ¹ . Prior clinical studies have identified adverse events ^(1,2) , and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments ⁷ , yet little is known […]
    Ersin Akinci
  • A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation May 28, 2020
    Empirical optimization of stem cell differentiation protocols is time consuming, is laborintensive, and typically does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet single-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA "barcodelets" to identify them on the basis of the treatments they receive. […]
    Grace Hui Ting Yeo
  • Author Correction: Predictable and precise template-free CRISPR editing of pathogenic variants February 16, 2019
    In this Article, a data processing error affected Fig. 3e and Extended Data Table 2; these errors have been corrected online.
    Max W Shen
  • Predictable and precise template-free CRISPR editing of pathogenic variants November 9, 2018
    Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with […]
    Max W Shen
  • A CRISPR view of gene regulation October 3, 2017
    Due to plummeting costs, whole genome sequencing of patients and cancers will soon become routine medical practice; however, we cannot currently predict how non-coding genotype affects cellular gene expression. Gene regulation research has recently been dominated by observational approaches that correlate chromatin state with regulatory function. These approaches are limited to the available genotypes and […]
    Budhaditya Banerjee
  • A CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer April 6, 2017
    CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant 'don't eat me' signal, we analyse […]
    Paola A Betancur
  • Self-Cloning CRISPR August 18, 2016
    CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the […]
    Mandana Arbab
  • A synergistic DNA logic predicts genome-wide chromatin accessibility July 27, 2016
    Enhancers and promoters commonly occur in accessible chromatin characterized by depleted nucleosome contact; however, it is unclear how chromatin accessibility is governed. We show that log-additive cis-acting DNA sequence features can predict chromatin accessibility at high spatial resolution. We develop a new type of high-dimensional machine learning model, the Synergistic Chromatin Model (SCM), which when […]
    Tatsunori Hashimoto
  • A distant trophoblast-specific enhancer controls HLA-G expression at the maternal-fetal interface April 15, 2016
    HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer […]
    Leonardo M R Ferreira
  • Cas9 Functionally Opens Chromatin April 1, 2016
    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility […]
    Amira A Barkal
  • High-throughput mapping of regulatory DNA January 26, 2016
    Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9-based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ∼40 kb of cis-regulatory genomic space and uses knock-in green […]
    Nisha Rajagopal
  • Cloning-free CRISPR November 4, 2015
    We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the […]
    Mandana Arbab
  • Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells June 26, 2015
    Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction […]
    Sissy E Wamaitha
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