...increase specificity, and decrease off-target effects Sniper-Cas9 - less off-target activity; compatible with...cleaving only one strand of target dsDNA. Off-target effects or off-target activity Cas9 cleavage at undesired...of partial homology throughout the genome, called off-targets, that can impact your experiment. There are...DSB within the target DNA, it’s unlikely that two off-target nicks will be generated close enough to cause... mutating specific amino acid residues to reduce off-target editing. Some mutations disrupt interactions...method, increased fidelity enzymes generate less off-target editing than wild type Cas9. Examples of increased...proofreading and discrimination evoCas9 - decrease off-target effects xCas9 3.7 - mutations in multiple ...

...cytosine base editors, which decrease the rate of off-target genome edits compared to rat APOBEC1. Now,...CRISPR/Cas9 in cells has advantages like reduced off-target activity and decreased immune response to ...ribonucleoproteins for transient expression. They replaced the tetraloop of the sgRNA scaffold with a com aptamer which...

...) the consequences to your experiment if there’s off-target expression.   Once you know that, you’re ready...tyrosine-hydroxylase (TH), can limit which cells express your gene. Off-target expression can still occur (Fig. 3) - for ... two astrocyte selective promoters demonstrating off-target expression in both oligodendrocytes (Olig2...promoter allows you to decide when to turn on, or turn off, gene expression by administering an inducing agent...  Finally, the risks of potential toxicity and/or off-target effects (e.g. recombinase-dependent vectors...Ubiquitous promoters  These often-strong promoters offer the most flexibility for expression in a large number...2002). Lack of an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term...

...on and off with light.  Xin hypothesized that she could make enzymes controllable by tethering them to...thereby occluding the enzyme active site and shutting off the enzyme. Later exposure to cyan light stopped ...

... in the rat APOBEC1 that decreases the number of off-target RNA edits by >390 fold and >3,800 fold in .... coli genome-wide CRISPR inhibition Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9...genome-wide CRISPR inhibition library Covalent tethering of DNA repair template to Cas9 Homology-directed...

... temporally control which genes are turned on or off in a given area and the can do so in a very specific...gene expression studies, which are evinced by the Tet system and visualization of single molecules via ... which allow you to easily and reversibly switch off and on your favourite signalling pathways (Figure...of the ribosome during translation, which knocks off all mRNA binding proteins as it travels along the...for Translation RNA Imaging by Coat protein Knock-off, is made up of three components: a GFP protein that...NLS-MS2-RFP) the reporter mRNA, which contains the tethering sites for the fluorescent proteins and an inducible...however, remain bound to the mRNA since they are tethered AFTER the stop codon. Thus a non-translated mRNA...

...PR, pLtetO, pLlacO Arabinose, Anhydrotetracycline, lactose, IPTG Richard Murray Plasmids based off of ... pdCas9-bacteria 44249 pTetO Anhydrotetracycline Stanley Qi Anhydrotetracycline inducible expression of... light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline inducible expression...pwtCas9-bacteria 44250 CRISPR Stanley Qi Anhydrotetracycline inducible expression of wild-type Cas9 from...

...some challenges still exist. Nucleases may cause off-target effects on regions of the genome with similar...either induce or repress Cre expression using TetON or TetOFF systems. So now hopefully creating a genetically... the cell can occur. Similar systems include tetracycline or doxycycline mediated activation of Cre to...

...designed to bind specific genes and turn them on or off, literally at the flip of a (blue) light switch. ...chromophores flavin-adenine dinucleotide and methyltetrahydrofolate, which are universally abundant. Second...apply LITEs for the transformation of neurons, she offers a tip: save yourself some time by making an AAV1...

... GFP-binding nanobodies, the project really took off. Unlike most antibodies, nanobodies, which occur ...DNA-binding domains, including the commonly used LexA and rTetR systems such that they activate gene expression ...advantages. T-DOGGs can be made drug-inducible using the rTetR system, but Cre-DOG lacks the toxicity that can ...

... on-target activity to siRNA, but with far fewer off-target effects! This characteristic probably explains... by DNMT3A or MQ1, and cytosine demethylation by Tet1. RNA Targeting: This page contains Cas13 constructs...

...receive your award, we’d love to see you show it off! To share your photo, tag @Addgene and #happydepositor... the world from the far-eastern islands,” said Tetsushi Sakuma, a lecturer at Hiroshima University who...

...detection methods lack spatiotemporal control or an “on/off switch.” This makes them great tools for providing...speed of labeling. BioID requires 6-24 hours for tetracycline induced expression of the BirA* fusion protein...Split-BioID, Schopp et al allowed 24 hours for tetracycline induced expression and biotinylation of interacting...

... underwent affinity screening to select for slow off-rate binders. The best candidates were validated ...where gRNA expression is induced by anhydrous tetracycline. Three new anti-CRISPRs were identified that...Janelia Farm GENIE project have arrived! We now offer several versions of GCaMP8 vectors in serotypes ...-mRuby from Liqun Luo’s lab expresses membrane tethered GFP to enhance axonal labeling and synaptophysin-tagged...

... Wheeldon FgH1tUTG 70183 Mammalian/Lentiviral H1-Tet none S. pyogenes Herold lentiGuide-Crimson 70683 ...Zhang pRS416-dCas9-Mxi1 + TetR + pRPR1(TetO)-NotI-gRNA 73796 Yeast pRPR1(TetO) yes, interfere S. pyogenes... 65006 Bacteria BsaI yes, interfere S. pyogenes Koffas BPK764 65767 Bacteria BsaI yes, cut S. pyogenes...yes, nick S. pyogenes Duchek pAC5-dual-dCas9VP48-sgTetO 48237 Mammalian BbsI yes, activate S. pyogenes ...Joung AAV:ITR-U6-sgRNA (Backbone) PCB-FlPO-WPRE-syntetisk pA-UTR 68347 Mammalian/AAV none S. pyogenes Elverlov-Jakobsen...62315 Yeast none S. pyogenes URA3 Lim, Qi pRPR1(1xTetO)_gRNA_handle_RPR1t 62966 Yeast HindIII none S. ... Mammalian/Lentiviral hU6 none S. pyogenes Puro Moffat lenti sgRNA(MS2)_puro backbone 73795 Mammalian/...

...learning and writing about science whenever he's not off the grid.   References Bischof J, Duffraisse M, Furger...interactions that uses split fluorescent proteins tethered to potential protein interaction partners. Unlike... form the complete FP because the PIPs they’re tethered to bring them into close proximity (Figure 1)....

... introduction of MerMAIDs, neurons can be turned off quickly and with high precision - a valuable optogenetics...latter experiments, such as transcription factors tethered to dCas9, require long time scales with constitutively...

...another, or their fluorescence can be switched on and off reversibly. This ability to modulate fluorescence...PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-FPS are better for whole cell imaging...Fluorescent proteins (FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate...UV-Violet Cyan/ Green >2,000 Medium 490/ 511 Kaede Tetramer UV-Violet Green/ Red ~2,000 High 572/ 580 tdEos...UV-Violet Green/ Red N/A High 573/ 584 KikGr Tetramer UV-Violet Green/ Red >2,000 High 583/ 593 mKikGR...Monomer Blue Dark/ Red 20 Low 572/ 610 FP595 Tetramer Green Dark/ Red 70 - 1000 Medium 590/ 600 ...non-invasive labelling, but in contrast, PA-FPs offer a reduced potential for photobleaching and phototoxicity...

...generate a knockout or edit. This can help reduce off-target activity. Deleting a large region of the genome...by L-arabinose and anhydrotetracycline (aTet), respectively. pMAZ-SK contains an aTet-inducible gRNA and...coli multiplexing plasmid is available from the Koffas lab. Scaling up your multiplexed gRNAs with Golden... on a separate plasmid. Multiplexing in E. coli Koffas Lab CRISPathBrick Multiplex Plasmid: This system...self-destruction" after induction with L-rhamnose and aTet. CRMAGE is much more efficient than traditional ...plasmids from this paper at Addgene. 3. Sakuma, Tetsushi, et al. “Multiplex genome engineering in human...

...binding partners does it have and how do they come off and on in a cellular environment? If these are pressing... nonfluorescent until they bind a 6-residue tetracysteine tag (sequence: CCPGCC) that can be inserted ...interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition, since these tags are so small...et al. "Hairpin structure of a biarsenical− tetracysteine motif determined by NMR spectroscopy." Journal...