... PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator peptide can increase...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene...separate gRNA expression plasmid to target the dCas9-activator to your specific locus. Plasmid Gene/Insert Promoter...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...

... PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator peptide can increase...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene...separate gRNA expression plasmid to target the dCas9-activator to your specific locus. Plasmid Gene/Insert Promoter... none S. pyogenes Yang pBUN6A11 OsU3 BsaI yes, activate S. pyogenes Bar Chen pBUN6I11 OsU3 BsaI yes, interfere.... pyogenes Bar Chen pHSN6A01 AtU6-26 BsaI yes, activate S. pyogenes Hyg Chen pHSN6I01 AtU6-26 BsaI yes...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...

... PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator peptide can increase...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene...separate gRNA expression plasmid to target the dCas9-activator to your specific locus. Plasmid Gene/Insert Promoter...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...Selectable Marker PI Publication Purify A catalytically inactive Cas9 (dCas9) can be used to purify a region of...

...) plasmids : Genome-wide screening of enhancer activity using a Firefly luciferase reporter. These plasmids... has increased stability and light output Sanjiv Sam Gambhir 104587 pHIV-iRFP720-E2A-Luc Firefly EF1α ...

...signal the presence of specific biomolecules, the activity of specific genes and cellular processes, or other...) High-performance calcium sensors for imaging activity in neuronal populations and microcompartments....GCaMP6f/m/s calcium sensors for imaging neural activity (Constitutive or Cre-dependent) Ultrasensitive...Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013 Jul 18;499(7458):295-300. Douglas ...expressing GCaMP6 and mRuby2 for imaging neural activity (GCaMP6f/m/s) (Constitutive or Cre-dependent) ...Cre-dependent) In vivo measurement of afferent activity with axon-specific calcium imaging. Nat Neurosci...

...fusion protein activates the expression of downstream genes. Multiple sgRNAs targeted to the same promoter ... either alone or fused to the transcriptional activator VP64. When directed to its target in gene promoters...promoter act synergistically to activate gene expression. The dCas9 protein serves as a negative control of the... the function of the VP64 activator and can also be used to inhibited gene expression via steric hindrance...These plasmids are described in: RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera...VP64 : Expresses inactivated S. pyogenes dCas9 (D10A, H840A) fused to VP64 transactivator domain in mammalian...below: ID Plasmid 47106 pcDNA-dCas9 : Expresses inactivated S. pyogenes dCas9 (D10A, H840A) in mammalian ...

... PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator peptide can increase...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory regions of your gene...separate gRNA expression plasmid to target the dCas9-activator to your specific locus. Plasmid Gene/Insert Promoter...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...Selectable Marker PI Publication Purify A catalytically inactive Cas9 (dCas9) can be used to purify a region of...Marker PI Publication Visualize A catalytically inactive Cas9 (dCas9) fused to a fluorescent protein (FP...

... scientists have developed constructs to activate/inactivate genes when Cre is present. By expressing ...addition of an exogenous ligand (e.g. tamoxifen) to activate Cre. One advantage of this system is tight temporal.... Cre may be expressed globally under a broadly active promoter like CAG, or expressed only in a subset...Pu 87694 pCAG-ERT2-PhoCl-Cre-PhoCl-ERT2 light-activated Cre cTnT Mammalian Campbell 89573 pCAG-iCre iCre...expression. Depending on the construct, Cre may activate or repress gene expression. Common construct types...Description Expression System PI 11578 pSico Cre activates shRNA expression, removal of EGFP; See also similar... Lentiviral Jacks 11584 Lox-Stop-Lox TOPO Cre activates gene of interest by removing Stop sequences; Puro...

...Cas9 based approach to knockout, repress, and activate gene targets. To accomplish this they have taken...sequence repression dCas9-VPR fusion for gene activation CRISPR/Cas9 Plasmid Variants Schematic of the...Plasmids 175571 and 175572 express dCas9-VPR for activation. Usage Guide sequences can be designed using ...sequence: Up to two guides can be expressed from the same plasmid by utilizing one of the dual guide shuttle...

...particular protein can activate expression from that element. When this activation requires a transcription... much about its function and/or alter cellular activity in a way that’s interesting from an applied perspective...more subtle control over protein expression and activity thereby limiting the use of cellular resources... a green light responsive sensor protein that activates expression of lacZ in response to green light....interact in a bacterial two-hybrid assay, they activate expression of the reporter gene. Plasmid ID Promoter...fusion protein in pACλCI-β-flap results in the activation of lacZ expression from FW102 OL2-62 . Wolfe ...promoter region cloned into pH3U3-zif268 (omega) activates expression of ura3 and his3. In bacterial strain...

...fluorophores are available in a range of colors activated by different wavelengths, allowing for easy differentiation...conjugated with HRP. Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane...antibody, followed by incubation and wash steps. Activate the conjugate and read the output via a plate ... antigen and the conjugate, typically HRP, is activated and its output measured. This method can be performed...secondary antibody is added. The conjugate is activated and the output is read. A standard curve with ...deposited into tubes or plate wells) by fluorescence activated cell sorting (FACS). Figure 7: Flow cytometry ...application Collect and lyse samples to make proteins available. Tissue samples may need additional processing...

...with less endogenous activation and reduced off target activation by ligands. PSAMs themselves are modified...engineered receptors that are only activated by otherwise inert drugs. Once activated, chemogenetic receptors hijack...First Structure–Activity Relationship Studies for Designer Receptors Exclusively Activated by Designer Drugs...DREADDs and PSAMs. Using DREADDs DREADDs DREADDS, or designer receptors exclusively activated by designer...provides a noninvasive method for manipulating cell activity, especially neuronal firing. What is chemogenetics...chemogenetics Because chemogenetic receptors are activated through administered drugs that must diffuse throughout... throughout the body, the onset of receptor activation is delayed and takes on the order of minutes. Studies...

...and pa= photoactivation) domains on either side of the active site of a constitutively active MEK1. This...localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates the ERK KTR and red...and gut cells of C. elegans causes the same phenotype. Activation of the Raf-MEK-ERK signaling pathway ...Protein Activity with GFP Resources at Addgene.org Find KTR reporter plasmids Find photoactivatable fluorescent...signaling networks can also be non-specifically activated and, like the game of telephone, often the signal...intermediate messengers. Optical control of kinase activity can provide greater spatiotemporal resolution ...system, light is used to turn “on” and “off” MEK1 activity by attaching two dimerizing photoswitchable Dronpa...

...delivery is the same in both cases, but activation depends on small molecule activation (chemogenetics)... Chemogenetics Timing Fast activation/deactivation Prolonged activation Targeting Further restricted...response or track the exact time of activation. Receptor activity is also drawn out as drugs can remain...offer the same level of control over timing. Because chemogenetics relies on chemical activation and the...neuroscience by granting targeted control over neuronal activity. While they serve similar purposes, both techniques...milliseconds, and can precisely link a neuron's activation to behaviors. Scientists continue to engineer...drugs throughout the body, the onset of receptor activation takes minutes. As a result, it is not possible...

...Suntag” Cas9 activator that recruits up to twenty copies of the p65-HSF1 transcriptional activator to each ...transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice...expression of the SPH activator paired with AAV sgRNA delivery make the SPH mouse an attractive tool to probe ...recent papers describe the development of CRISPR activation (CRISPRa) tools for neuroscience, including transgenic...neuroscience research. What is CRISPR-based activation? To summarize, CRISPRa uses a nuclease-dead variant...and engineered combinations of transcriptional activators to induce gene transcription at genomic sites... vitro settings due to the large size of Cas9 activators, which largely precludes delivery by adeno-associated...

... described the first light-activated tool for controlling neuronal activity, channelrhodopsin-2 (ChR2)...exposed to blue light, this channel activates neuronal activity by opening and allowing positively charged...cellular activity with exquisite precision using only light. Optogenetics, or light activated tools, can...either activate or inhibit a specific receptor or pathway.  Light-gated ion channels Light activated ion ...ChR2) activation by light showing ions flowing with the concentration gradient, and of activation inhibitory... to prevent activation of both tools with each light source used.   Channel and activation kinetics Some...ChR2), a blue light-activated cation channel, from the archaebacteria Chlamydomonas reinhardtii. When exposed...

...way to shut off Cas’s activity. The concern is that the longer Cas remains active in a cell, the greater...switch and turn off CRISPR activity. The ability to turn off CRISPR activity is an important step towards...Cas9 activity in HEK293 by reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr ... Cas proteins can be used for genome editing, activating gene expression, repressing gene expression, ...edits to occur. Although methods to switch on Cas activity using light or drugs have been developed, the ...proteins are highly diverse but most block CRISPR activity one of three ways: Inhibit DNA binding Example... Anti-CRISPR proteins (Acr's) can block CRISPR activity in many ways such as inhibiting crRNA loading,...

... at specific loci. Activate or Repress : dCas9 fused to transcriptional activators or repressors can modulate...processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic...for gene knockout, as well as transcriptional activation or repression. Combined with the power of next-generation...gRNAs: Truncated gRNAs display less off-target activity. High fidelity Cas9s: Three high fidelity Cas9...eSpCas9, and HypaCas9 display very low off-target activity due to rationally designed mutations. Researchers...CRISPR-Mediated Control of Gene Repression and Activation. Cell . 159(3):647-6. PMID: 25307932 Gilbert ...targeting multiple loci can be easily expressed in the same cell or organism. The ability to precisely target...

...transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation (TA) domains...discovery in the field of eukaryotic transcriptional activation and cell biology. During this period, proteins...expressed as fusions that retain their individual activities (1). Researchers also discovered the modular ...domains retain their individual activities when separated (2), and that DBD and TAs from different systems ...protein shown is still able to bind to the upstream activation sequence (UAS) of its target gene, in this case...of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists took advantage...commonly referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the...

... are always active and thus referred to as constitutive promoters. Others are only active under specific...Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the activator protein...concentrations are low, pADH1 displays low promoter activity (~20% activity of the strong yeast promoter pTEF). As ...or when that promoter is active. In this way, you can interrogate the activity of uncharacterized promoters...or VP16 activation domain results in chimeric GAL80-repressible and GAL80-insensitive activators of lexAop...introduced the second generation QF2 activators. These activators solve the problem of broad QF expression-induced...tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional activation domain VP16 taken...