... BbsI yes, activate, N-terminal S. pyogenes Zhang PX856 62888 Mammalian BbsI yes, activate, C-terminal...Bacteria BsaI yes, activate S. pyogenes Marraffini pWJ68 46571 Bacteria BsaI yes, activate S. pyogenes Marraffini...yes, activate S. pyogenes Luikart pRubiG-T2A-Cas9 75348 Mammalian/Retroviral from pXL yes, activate S. ... none S. pyogenes Boxem lentiSAMv2 75112 Mammalian/Lentiviral U6 yes, activate S. pyogenes Blast Zhang...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...-in-one vector, but their function (cut, nick, activate, interfere, etc.) is limited to that of the Cas...Cas9. We have plasmids with Cas9 that can cut, activate, interfere, or nick. Selection , such as Puromycin...

... 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. ID Plasmid Gene/Insert...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. ID Plasmid Gene/Insert...S. pyogenes Yang 50579 pBUN6A11 OsU3 BsaI yes, activate S. pyogenes Bar Chen 50580 pBUN6I11 OsU3 BsaI ...pyogenes Bar Chen 50586 pHSN6A01 AtU6-26 BsaI yes, activate S. pyogenes Hyg Chen 50587 pHSN6I01 AtU6-26 BsaI...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. ID Plasmid Gene/Insert...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...

...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...

... 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. ID Plasmid Gene/Insert...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...

...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...

.../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 Figure 1: Schematic...Plasmids 175571 and 175572 express dCas9-VPR for activation. Usage Guide sequences can be designed using ... a unique enhancer and either Ascl1 or Neurog2 activity . Development, 148(12). doi: 10.1242/dev.199399...sequence: Up to two guides can be expressed from the same plasmid by utilizing one of the dual guide shuttle...

...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...

... 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...

... 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,...

...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...

...promoter is inactive because the activator protein cannot bind. After an inducer binds to the activator protein...direct activation. In this system, the activator rtTA (reverse tetracycline-controlled transactivator) is... are always active and thus referred to as constitutive promoters. Others are only active under specific...OFF state, the promoter is inactive because a bound repressor protein actively prevents transcription. Once...protein, the activator protein can bind to the promoter, turning it ON and initiating transcription.     ...derivatives serve as inducing agents to allow promoter activation. One of the most commonly used prokaryotic promoters...repressor (lacI protein) for transcription to be activated. In the presence of lactose or lactose analog ...

... optically active saCas9 protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR...proteins heterodimerize and move the activator into position to activate gene transcription. The researchers...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64...can interfere with activity. The resulting system was coined as CRISPR–Cas9 activity switching via a novel...generate a photoactivatable nickase and a photoactivatable repressor (dCas9). The activity of all variants...Shining light on transcriptional activation using dCas9 Initial photoactivatable CRISPR systems published in...genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase...

...approach can also be used for activation of specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP...endogenous gene activation. This system works by bringing together an anchor protein and activator protein to...portion of CRY2) fused to the p65 activation domain, which activates transcription of downstream genes...the targeted activation of endogenous genes and detail the time course of gene activation using this system...2015 Feb 23. A novel dCas9 activator capable of unprecedented activation of target genes A group of researchers...fused to a VP64 activator domain and targeted toward endogenous promoters to activate transcription. Chavez...synergistic effect on target gene activation. The novel dCas9-VPR activator is capable of enhancing target...

...balance maximizing on-target activity while minimizing off-target activity, which sounds obvious but can...to use a less-active gRNA that targets a truly unique site in the genome, or a more-active gRNA with one...that your gRNA sequence is designed to be highly active and reduce off targets. For CRISPRa and CRISPRi...to generate the desired edit. The wrench: Gene activation and inhibition by CRISPRa and CRISPRi Finally...expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition) technologies – a nuclease-dead...100nt window downstream of the TSS gives the most activity. Thus, a given gene will only have a dozen or ...features to nominate gRNAs that are likely to be active, not only for SpCas9 but also for some other Cas...