Jonathan S Weissman

Harvard Medical School, Boston, Massachusetts, United States

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Publications (309)

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    [Show abstract] [Hide abstract] ABSTRACT: Background Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that can regulate molecular and cellular processes in brain development and disease. LncRNAs exhibit cell type- and tissue-specific expression, but little is known about the expression and function of lncRNAs in the developing human brain. Furthermore, it has been unclear whether lncRNAs are highly expressed in subsets of cells within tissues, despite appearing lowly expressed in bulk populations. Results We use strand-specific RNA-seq to deeply profile lncRNAs from polyadenylated and total RNA obtained from human neocortex at different stages of development, and we apply this reference to analyze the transcriptomes of single cells. While lncRNAs are generally detected at low levels in bulk tissues, single-cell transcriptomics of hundreds of neocortex cells reveal that many lncRNAs are abundantly expressed in individual cells and are cell type-specific. Notably, LOC646329 is a lncRNA enriched in single radial glia cells but is detected at low abundance in tissues. CRISPRi knockdown of LOC646329 indicates that this lncRNA regulates cell proliferation. Conclusion The discrete and abundant expression of lncRNAs among individual cells has important implications for both their biological function and utility for distinguishing neural cell types. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0932-1) contains supplementary material, which is available to authorized users.
    Full-text Article · Dec 2016 · Genome biology
  • Article · Jul 2016 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: The Cancer Target Discovery and Development (CTD2) Network was established to accelerate the transformation of "Big Data" into novel pharmacological targets, lead compounds, and biomarkers for rapid translation into improved patient outcomes. It rapidly became clear in this collaborative network that a key central issue was to define what constitutes sufficient computational or experimental evidence to support a biologically or clinically relevant finding. This manuscript represents a first attempt to delineate the challenges of supporting and confirming discoveries arising from the systematic analysis of large-scale data resources in a collaborative work environment and to provide a framework that would begin a community discussion to resolve these challenges. The Network implemented a multi-Tier framework designed to substantiate the biological and biomedical relevance as well as the reproducibility of data and insights resulting from its collaborative activities. The same approach can be used by the broad scientific community to drive development of novel therapeutic and biomarker strategies for cancer.
    Article · Jul 2016 · Molecular Cancer Research
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    [Show abstract] [Hide abstract] ABSTRACT: Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.
    Full-text Article · Jul 2016 · Nature Communications
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    [Show abstract] [Hide abstract] ABSTRACT: Supplementary Figures 1-8, Supplementary Table 1, Supplementary Methods and Supplementary Reference
    Full-text Dataset · Jul 2016
  • Yi-Chang Liu · Danica Galonić Fujimori · Jonathan S. Weissman
    [Show abstract] [Hide abstract] ABSTRACT: Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p-Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae We reconstitute this step in vitro with Htm1p-Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p-Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p-Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.
    Article · Jun 2016 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.
    Article · Jun 2016 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.
    Article · Jun 2016 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.
    Article · May 2016 · Cell
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    Full-text Dataset · Apr 2016
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    Full-text Dataset · Apr 2016
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    [Show abstract] [Hide abstract] ABSTRACT: In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
    Full-text Article · Mar 2016 · Nature Communications
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    [Show abstract] [Hide abstract] ABSTRACT: Supplementary Figures 1-3 and Supplementary Tables 1-6
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: Movie of mouse IMCD3 cells expressing either IFT20::GFP (left) or IFT20::GFP11x7 + GFP1-10 (right) taken by TIRF microscopy. Images were acquired every 50 ms.
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: Movie of Drosophila S2 cells expressing either GFP::β-tubulin (left), GFP11x1::β-tubulin (middle) or GFP11x7::β-tubulin (right) taken by confocal microscopy. Images were acquired every 400 ms.
    Full-text Dataset · Mar 2016
  • Max A Horlbeck · Lea B Witkowsky · Benjamin Guglielmi · [...] · Jonathan S Weissman
    [Show abstract] [Hide abstract] ABSTRACT: ELife digest Many bacteria have a type of immune system known as CRISPR that can target and cut foreign DNA to protect it against viruses. Recently, the CRISPR system was adapted to allow scientists to easily manipulate the genome of humans and many other organisms. However, unlike the loosely organized DNA found in bacteria, the DNA that makes up the human genome is tightly packed and wrapped around complexes of proteins to form structures called nucleosomes. It was not clear whether the CRISPR system was able to effectively target the stretches of DNA in a nucleosome. In 2013, researchers developed a modified version of CRISPR, known as CRISPR interference, to block gene activity and in 2014 used it to systematically repress many of the genes in the human genome. Now, Horlbeck, Witkowsky et al. – who include several of the researchers from the 2014 work – have analyzed existing data for a specific type of human cell grown in the laboratory and found that CRISPR interference activity was strongest in certain areas around the start of each gene. However, CRISPR interference was much weaker in other areas of genes that coincided well with stretches of DNA that are known to often be bound by nucleosomes. Nucleosomes also appeared to block CRISPR editing, although the effects were less pronounced. Horlbeck, Witkowsky et al. then directly tested whether nucleosomes could prevent the CRISPR system from binding or modifying the DNA. When the individual components were mixed in test tubes, the CRISPR system could readily target “naked” DNA. However, it could not access nucleosome-bound DNA, unless an enzyme that can move nucleosomes along the DNA in the human genome was also added to the mix. These findings suggest one way that CRISPR can manipulate much of the human genome despite the widespread presence of nucleosomes. Future work will now aim to develop computational methods that take the positions of nucleosomes into account when picking DNA sites to target with CRISPR. DOI: http://dx.doi.org/10.7554/eLife.12677.002
    Article · Mar 2016 · eLife Sciences
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    Max A Horlbeck · Lea B Witkowsky · Benjamin Guglielmi · [...] · Jonathan S Weissman
    [Show abstract] [Hide abstract] ABSTRACT: Table S2. Ricin tiling library sgRNA annotations, phenotype scores, and target site MNase signal, related to Figure 2. DOI: http://dx.doi.org/10.7554/eLife.12677.014
    Full-text Dataset · Mar 2016
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    Max A Horlbeck · Lea B Witkowsky · Benjamin Guglielmi · [...] · Jonathan S Weissman
    [Show abstract] [Hide abstract] ABSTRACT: Table S1. CRISPRi sgRNA annotations, activity scores, and target site MNase signal, related to Figure 1. DOI: http://dx.doi.org/10.7554/eLife.12677.013
    Full-text Dataset · Mar 2016
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    [Show abstract] [Hide abstract] ABSTRACT: Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.
    Full-text Article · Mar 2016 · Cell Stem Cell
  • Luke A. Gilbert · Max A. Horlbeck · Jacqueline Villalta · [...] · Jonathan S. Weissman
    Article · Feb 2016 · Cancer Research

Publication Stats

37k Citations

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Institutions

  • 2012
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2002-2011
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2003-2010
    • University of California, San Francisco
      • • Department of Cellular and Molecular Pharmacology
      • • Department of Biochemistry and Biophysics
      San Francisco, California, United States