Showing papers by "Phillip A. Sharp published in 2014"

CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling

Abstract: CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated KrasG12D mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Abstract: Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.

Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

Abstract: We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

Abstract: We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

CRISPR-mediated direct mutation of cancer genes in the mouse liver

Abstract: The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

CRISPR-mediated direct mutation of cancer genes in the mouse liver

Abstract: The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre–LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre–loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

LincRNA-p21 Activates p21 In cis to Promote Polycomb Target Gene Expression and to Enforce the G1/S Checkpoint

Abstract: The p53-regulated long noncoding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function, we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a coactivator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, a defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted coactivator for p53-mediated p21 expression.

Target specificity of the CRISPR-Cas9 system

Abstract: The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.

Rbfox2 controls autoregulation in RNA-binding protein networks

Abstract: The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

Building Robust Transcriptomes with Master Splicing Factors

Abstract: Coherent splicing networks arise from many discrete splicing decisions regulated in unison. Here, we examine the properties of robust, context-specific splicing networks. We propose that a subset of key splicing regulators, or "master splicing factors," respond to environmental cues to establish and maintain tissue transcriptomes during development.

Argonaute-Bound Small RNAs from Promoter-Proximal RNA Polymerase II

Abstract: Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic stem cell system to express a single epitope-tagged Ago protein family member in an inducible manner. Here, we report the small RNA profile of Ago-deficient cells and show that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, we identified an Ago-dependent class of noncanonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation.

Global microRNA depletion suppresses tumor angiogenesis

Abstract: MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3' untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.

Quercetin Inhibits Intestinal Iron Absorption and Ferroportin Transporter Expression In Vivo and In Vitro

Abstract: Balancing systemic iron levels within narrow limits is critical for maintaining human health. There are no known pathways to eliminate excess iron from the body and therefore iron homeostasis is maintained by modifying dietary absorption so that it matches daily obligatory losses. Several dietary factors can modify iron absorption. Polyphenols are plentiful in human diet and many compounds, including quercetin – the most abundant dietary polyphenol – are potent iron chelators. The aim of this study was to investigate the acute and longer-term effects of quercetin on intestinal iron metabolism. Acute exposure of rat duodenal mucosa to quercetin increased apical iron uptake but decreased subsequent basolateral iron efflux into the circulation. Quercetin binds iron between its 3-hydroxyl and 4-carbonyl groups and methylation of the 3-hydroxyl group negated both the increase in apical uptake and the inhibition of basolateral iron release, suggesting that the acute effects of quercetin on iron transport were due to iron chelation. In longer-term studies, rats were administered quercetin by a single gavage and iron transporter expression measured 18 h later. Duodenal FPN expression was decreased in quercetin-treated rats. This effect was recapitulated in Caco-2 cells exposed to quercetin for 18 h. Reporter assays in Caco-2 cells indicated that repression of FPN by quercetin was not a transcriptional event but might be mediated by miRNA interaction with the FPN 3′UTR. Our study highlights a novel mechanism for the regulation of iron bioavailability by dietary polyphenols. Potentially, diets rich in polyphenols might be beneficial for patients groups at risk of iron loading by limiting the rate of intestinal iron absorption.

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

Abstract: Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ∼3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo.

Target specificity of the CRISPR-Cas9 system

Abstract: National Institutes of Health (U.S.) (United States Public Health Service Grant RO1-GM34277)

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

Abstract: Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.

Meeting Global Challenges

Abstract: ![Figure][1] CREDIT: COURTESY OF PHILLIP A. SHARP; COLELLA PHOTOGRAPHY Every major challenge of modern life, such as ensuring energy, health, water, or food security in a sustainable world with a predicted nine billion inhabitants, has complex science and technology (S&T) components and is global in character, even though its expression often involves national idiosyncrasies. Searching for solutions requires that the scientific community operate in fundamentally new ways. How to deal with these complex global issues is a major focus of this year's annual meeting of the American Association for the Advancement of Science (AAAS), whose theme is Meeting Global Challenges: Discovery and Innovation . Developing effective solutions requires converging approaches, such as the integration of knowledge from the life, physical, social, and economic sciences and engineering. Moreover, the search for solutions needs to draw upon the talents and innovative ideas of scientists, engineers, and societal leaders worldwide to overcome traditional and nationalistic paradigms that have so far been inadequate to meeting these challenges. Unfortunately, neither S&T funders nor performing institutions are well organized, nor are members of the S&T community well trained for working in these ways. That will have to change. ![Figure][1] CREDIT: KEMALBAS/ISTOCKPHOTO.COM Education and training programs must be developed in what has come to be called “convergence science”: the integration of life, physical, and engineering sciences, so that S&T practitioners have a knowledge and experience base to participate in the kinds of integrated scientific efforts that are needed. More opportunities are also needed for scientists to collaborate in international settings and participate in global science projects during their training years, so that international collaboration becomes a more natural part of the scientific culture. Research-performing and training institutions, such as universities and research institutes, have critical roles to play. Not only is it essential that they develop appropriate training programs and help stimulate multidisciplinary international collaborations, but they also need to reconsider some traditional incentive structures. Performing institutions should encourage and reward scientists and engineers for their work in large multidisciplinary, multinational teams. Institutions should also help provide the resources necessary to nurture these types of collaborations. Fortunately, there is increasing recognition among some science funders of the need for enhanced mechanisms for funding science in a global, multinational fashion. The Heads of International Research Organizations, an aggregation of health research funders from around the world, meets regularly, with the goal of increasing consistency in policies across countries and facilitating global cooperation to tackle major health issues. The Global Research Council has brought together the heads of 70 basic science funding agencies from diverse countries and is working toward harmonizing policies on topics such as research integrity, peer review, and access to data and publications. AAAS has convened a variety of groups to work on bringing greater coherence and consistency to policies and practices across countries, primarily at the regional level, such as in East Africa and the Asia-Pacific region. But harmonizing science policies across countries and their funding agencies and taking advantage of collaborative opportunities will not be sufficient. The work products from these multinational, multidisciplinary teams must be rapidly translated into practical solutions. To facilitate that translation, scientists and engineers must engage with business, cultural, and political leaders. Further, the adoption of national policies that address global challenges is dependent on support by an informed citizenry and public debate stimulated by discussions with policy-makers around the world. The international multidisciplinary AAAS Annual Meeting provides forums for such discussions and should produce an array of practical strategies and solutions to bring the full power of science to bear on world challenges. [1]: pending:yes

Meeting global challenges: Discovery and innovation through convergence

Abstract: Our awareness of the global nature of major problems facing our planet is relatively new and demands global responses for which neither the scientific community nor the general public is well prepared. To meet such global challenges requires the engagement of people and their leaders from diverse cultures and experiences. While many sectors of society must become involved, the scientific community has a special role in preparing for these challenges. It is impossible to consider here all of the roles of scientists and engineers in this transformation, whether advancing knowledge of nature, translating new insights into innovations, or educating future generations. But few would disagree that in the long term, discovery and innovation are central to effectively meeting these challenges. Yet it is often difficult to link discovery and innovation to their impact because of the years of development required before a discovery matures to a level that can be adopted on a global scale. I highlight below how some organizations are planning to accelerate the transition from discovery to innovation to address some of the great challenges facing society. As an illustration, I discuss the history of the transition from discovery to innovation at the molecular level in life sciences. At the end, I comment on how further convergence of physical, mathematical, engineering, and social sciences with life sciences will accelerate innovation.

Micromilling enhances iron bioaccessibility from wholegrain wheat.

Abstract: Cereals constitute important sources of iron in human diet; however, much of the iron in wheat is lost during processing for the production of white flour. This study employed novel food processing techniques to increase the bioaccessibility of naturally occurring iron in wheat. Iron was localized in wheat by Perl’s Prussian blue staining. Soluble iron from digested wheat flour was measured by a ferrozine spectrophotometric assay. Iron bioaccessibility was determined using an in vitro simulated peptic–pancreatic digestion, followed by measurement of ferritin (a surrogate marker for iron absorption) in Caco-2 cells. Light microscopy revealed that iron in wheat was encapsulated in cells of the aleurone layer and remained intact after in vivo digestion and passage through the gastrointestinal tract. The solubility of iron in wholegrain wheat and in purified wheat aleurone increased significantly after enzymatic digestion with Driselase, and following mechanical disruption using micromilling. Furthermore, fol...

Alginate inhibits iron absorption from ferrous gluconate in a randomized controlled trial and reduces iron uptake into Caco-2 cells.

Abstract: Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n=15) were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p=0.003). Sub-group B (n=9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p=0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p=0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p=0.009) and 35% (p=0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p,0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification. Trial Registration: ClinicalTrials.gov NCT01528644

Corrigendum: Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

Abstract: Corrigendum: Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype