CRISPR transcriptional repression devices and layered circuits in mammalian cells

A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50 GM098792)Korea (South). Ministry of Science, Information and Communication Technolgy. Intelligent Synthetic Biology Center of Global Frontier Project (2013M3A6A8073557

Cas9 gRNA engineering for genome editing, activation and repression

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.National Human Genome Research Institute (U.S.) (P50 HG005550)United States. Department of Energy (DE-FG02-02ER63445)Wyss Institute for Biologically Inspired EngineeringUnited States. Army Research Office (DARPA W911NF-11-2-0054)National Science Foundation (U.S.)United States. National Institutes of Health (5R01CA155320-04)United States. National Institutes of Health (P50 GM098792)National Cancer Institute (U.S.) (5T32CA009216-34)Massachusetts Institute of Technology. Department of Biological EngineeringHarvard Medical School. Department of GeneticsDefense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006

Bioreactor technologies to support liver function in vitro

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.National Institutes of Health (U.S.) (R01 EB010246)National Institutes of Health (U.S.) (P50-GM068762-08)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109)5UH2TR000496-02National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039

Cytokine secretion by hepatocytes, KCs, LSECs and HSCs after isolation from the same liver and in response to low levels of LPS.

<p>Liver cells were freshly isolated on density gradient followed by cell sorting and stimulated with LPS (1ng/mL LPS, black bars or 100ng/mL LPS, hatched bars). Cytokine secretion was measured in the same supernatant with a multiplex assay, run in triplicates. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between basal LPS stimulation (1ng/mL) and higher LPS stimulation (100ng/mL) are indicated. Lower panel: bright field images of cells right after isolation (Hepatocytes, LSECs, KCs). Images of HSCs at higher resolution show the retinol droplets at Day 0 and the typical shape of the activated stellate cells after 4 days in culture.</p

Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.

<p>Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.</p

Hepatic stellate cells are the major source of CXCL1, as shown by both quantification of secretion and <i>in situ</i> localization.

<p><b>(A)</b> Quantification of CXCL1 secretion in enriched fractions of hepatocytes, KCs, LSECs and HSCs, freshly isolated and stimulated <i>in vitro</i> with LPS (1 ng/mL LPS, black squares) during 24 hours. Data are representative of three separate experiments with six mice in each group; ^<b>#</b><i>P</i><.05. <b>(B)</b> <i>In-situ</i> localization of CXCL1 in the liver. Immunofluorescent detection for CXCL1 (red) and liver cells nuclei (blue) for nuclei first shows CXCL1 expression in the sinusoids throughout liver parenchyma. <b>(C)</b> Higher resolution shows that CXCL1 (red) is expressed by sub-endothelial cells, which also store retinol droplets in separate compartments, as shown by CRBP1 staining (green). The Cellular Retinol Binding Protein-1 (CRBP-1) is the best marker to detect simultaneously both resting (Glial Fibrillary Acidic Protein, GFAP+) and activated (α-Smooth Muscle Actin, αSMA+) stellate cells <i>in situ</i>. Alexa Fluor-546-CXCL1 (red) staining does not colocalize either with Tie2-GFP in LSECs (green, <i>upper panel</i>), or F4/80 in KCs (blue, <i>middle panel</i>), but with AlexaFluor-488-CRBP1 (green, <i>lower panel</i>), staining both resting and activated HSCs. TOPRO3 was used for nuclei vizualisation.</p

Neutrophils migrate in response to CXCL1 secretion following TLR4 activation in hepatic stellate cells.

<p><b>(A)</b> Schematic representation of the neutrophil chemotaxis assay. <b>(B)</b> Quantification of neutrophil migration in response to secretory WT or TLR4 deficient stellate cells. WT stellate cells were treated (WT HSC + anti CXCL1) or not with anti-CXCL1 antibody. As for internal positive control, the migration of neutrophils towards TLR4 deficient stellate cells supplemented with CXCL1 protein (TLR4 HSC + CXCL1) and with CXCL1 protein only (CXCL1) was quantified in only one experiment. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between WT HSCs and TLR4 deficient HSCs, as well as between WT HSCs treated or not with anti-CXCL1, are indicated.</p

Decrease of CXCL1 message and neutrophil counts in TLR4 deficient liver and after antibiotic treatment.

<p><b>(A)</b> CXCL1 expression in the total liver as analyzed by microarrays. Mean values were obtained from three Genechips for three WT and three TLR4 deficient mice. Statistically significant differences between WT and TLR4 deficient mice are indicated by an asterisk, *<i>P</i><.05, Student <i>t</i> test. (<b>B)</b> CXCL1 expression measured by quantitative RT-PCR. The relative quantity of CXCL1 mRNA in the total liver of WT and TLR4 deficient mice is indicated (*<i>P</i><.01). <b>(C)</b> Relative expression of CXCL1 in the liver from untreated or antibiotic-treated (ABT) WT mice and TLR4 deficient mice; *<i>P</i><.01 <b>(D)</b> Neutrophils counts in the total liver. CD11+ Gr1^high TCR- cells among total live leukocytes isolated from WT and TLR4 deficient liver. In Fig 1B, 1C and 1D, data are representative of five separate experiments with six WT mice (treated or not with antibiotics) and five TLR4 mice; ^<b>#</b><i>P</i><.05; unpaired Mann -Whitney test.</p