...sgRNA2-2x35S-5'UTR::Cas9::NOST-AtU6p::sgRNA1 : Level 2 construct....CRISPR Protocols gRNA Design Tools CRISPR Blog Posts A CRISPR/Cas system designed and optimized for [email protected] . These plasmids are described in: Targeted mutagenesis in the model plant Nicotiana...in the plant tissue. 46968 pICSL01009::AtU6p : Encodes the Arabidopsis U6 promoter in a level 0 vector...vector for plant gene expression (S. Marillonnet, described in Weber et al. (PLoS One 6:e16765, 2011). 49770...

...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts CRISPR/Cas nickase mutants...Selectable Marker PI Publication Yeast ID Plasmid Description Gene/Insert Promoter Selectable Marker PI Publication...

...2007 ) in mammalian cells or zebrafish). pMG10 (a '“2-in-1” ZFN cassette for expression of both a “+” and...This page lists plasmid vectors designed to express nucleases in different cell types. Zinc finger arrays...Expression Vectors This page lists plasmid vectors designed to express nucleases in different cell types. ...zebrafish (can be used as a substitute for pMLM335 described in Foley et al., PLoS One 2009) pMLM292 (for expression...zebrafish (can be used as a substitute for pMLM336 described in Foley et al., PLoS One 2009) pMLM800 (for expression...Methods 2011 ) in mammalian cells or zebrafish as described by Moore et al. PLoS One 2012 and Cade et al.,...

...vectors include the germline expression compatible tbb-2 3’UTR. For both the eft-3 and hsp-16.48 vectors we...also like... CRISPR Guide CRISPR Protocols gRNA Design Tools CRISPR Blog Posts We adapted the S. pyogenes...production of RNA from the T7 promoter, while pMB70 is designed for in vivo transcrition controlled by the regulatory...into BsaI digested vector. These plasmids are described in: CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative... is described in more detail in the Multiplex Assays section below. The following table describes the ... Science Guides Antibody Guide Antibody Guide View Addgene's Antibody...

...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, PAM requirements...Protocols gRNA Design Tools CRISPR Blog Posts The table below lists gRNA plasmids designed to target various...pyogenes , S. aureus etc.) was this gRNA sequence designed for? A given gRNA sequence may only be compatible...unpublished plasmids containing gRNA sequences designed to be used with any CRISPR nuclease and function...

...196208 GB1-A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep...196211 GB1-A11(2-196, G175R)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196212 GB1-A11(2-196, G189E...8661 p4455 FLAG-hPLIC-2 UBQLN2 Flag CMV ALS Peter Howley 8662 p4456 FLAG-hPLIC-2 NTF UBQLN2 Flag CMV ALS...Pietro De Camilli 194005 VPS13D1-1576-(Y1253S) VPS13D GFP CMV Spinocerebellar Ataxia Pietro De Camilli... Lalit Deshmukh 196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196211 ... ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196214 GB1... ALS Lalit Deshmukh 196216 GB1-A11(2-68)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196694 mBFP-APP-mGFP...

...optogenetic tool is the widely used Channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii . ChR2 is a ...many different types of proteins. For a full description (including peak response spectra) of different...preparations of many optogenetics plasmids. ID Plasmid Description Gene/Insert Vector Type Promoter PI Publication...transcription", "Hahn", or other terms. ID Plasmid Description Gene/Insert Vector Type Promoter PI Publication...

... funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777372....proteomic interactions represent the framework that provides biological context. Assays monitoring impedance...sub-cloning into a wide range of Gateway-compatible pDEST vectors. ID Plasmid Gene/Insert Industry PI Pooled...solute carrier family of proteins. Library ID PI Description Human SLC Activation Library 132561 Superti-Furga...

...the cells will still appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity...I got clear bands using the conditions above. 2. Design your primers so that the digestion site isn’t ...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing it to the undigested control...

...the use of pAdEasy-2 can increase the capacity of the adenoviral vector. pAdEasy-2 does not contain the...Adenovirus packaging plasmid that lacks E1 and E3 pAdEasy-2 Adenovirus packaging plasmid that lacks E1, E3, and...911E4 cells Cell line compatible with the pAdEasy-2 packaging plasmid; Supplies viral protein E4 E1 Supplied... Science Guides Adenoviral Guide Adenoviral Guide Components Vector...be harvested about 7-10 days later. Vogelstein designed multiple shuttle vectors for different purposes...

...AAV vectors for trial purposes in serotypes AAV1, 2, 5, 8, 9 and retrograde Engineered Serotypes Retrograde...: AAV Addgene's viral vector packaging service provides high-quality and high-titer viral preparations...

...receptor fused to GFP for studying cell ablation. 2 Jessell , Azim 45580 pAAV-flex-taCasp3-TEVp EF1a-driven...Cre-dependent Cre-dependent, bicistronic expression of designer pro-taCasp3 and TEVp for studying cell ablation...

...gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh...Protocols, 2016 . Yamamoto Tagging Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts The CRISPR system has been...plasmids and protocols needed for using them, are described below. Read our CRISPR 101 post for more information...information about HDR/repair template design. How to use CRISPR to tag your gene of interest Mendenhall and...interest is currently unavailable, you will need to design and clone in a gRNA(s) to guide the Cas9 protein...protein to your target sequence, as well as design and clone in the homology arms for the donor plasmid. The...

...silencing, and desynchronization of neural activity, with single-spike temporal resolution. PLoS One. 2(3):e299...influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted optogenetic tool,... (Phy) interaction 660 Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein ...interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control...transcription effectors OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated...comparative analysis of microbial opsins. Nat Methods. 9(2):159-72. PMID 22179551 Saunders A, Johnson CA, Sabatini...

... Hacohen N, Hahn WC, Lander ES, Sabatini DM, Root DE. Cell. 2006. 124:1283-1298. PubMed . A versatile ...MO, Trono D, Aebischer P. Nat Methods. 2006. Feb;3(2):109-16. PubMed . In vivo RNA interference demonstrates...site. Find resources for more information on shRNA design and delivery. Constitutive Lentiviral shRNA Lentiviral...for constitutive shRNA expression ID Plasmid Description PI 10878 pLKO.1 ‐ TRC cloning vector shRNA expression... included in the vector to monitor expression; Designed for inducing RNAi in a wide range of cell types...conditional (Cre-lox) shRNA expression ID Plasmid Description PI 11578 pSico Cre addition causes EGFP to be...for Tet-regulated shRNA expression ID Plasmid Description Tet on or off PI 11643 pLVCT-tTR-KRAB 2nd generation...

...dCas9VP64 and sgRNA from separate promoters. Table 2. pmax dCas9 Activator Expression Plasmids ID Plasmid...Table 4. Gateway Destination Vector ID Plasmid 48222 pAC90-pmax-DEST : Gateway Destination vector derived...the dCas9 protein provides the transactivation activity, the sgRNA component provides the specificity and...and can be transferred to Gateway destination vectors (e.g., pmax-DEST [48222] and (Link opens in a new...also like... CRISPR Guide CRISPR Protocols gRNA Design Tools CRISPR Blog Posts CRISPR-on is an RNA-guided... and is designed to target promoters or enhancers of genes of interest. Different flavors of activators...new window) Lifetechnologies Gateway Destination Vectors ) to adapt to different systems by LR clonase ...

... greater toxicity than guides that had zero mismatch off-targets. Guides with 2-bp mismatch off-targets...tolerated than mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target activity...PMID: 28474669. Pubmed Central PMCID: PMC5424143. 2. Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide...safe-targeting guides vs. non-targeting guides on screen analysis and hit calling. 5,644 non-targeting guides and...safe-targeting guides were included in the library. Safe-targeting guides were designed to target genomic...short guides had reduced toxicity compared to full-length guides. Reduced toxicity of short guides could...CRISPR screen design. Unanswered questions about genome-wide CRISPR/Cas9 screen design Are non-targeting...

...Figure 2. PSAM4-based LGICs, their effect, and outcome in neurons. Please see the text or table 2 for details... details. Table 2. Descriptions of PSAMs and their activity in neurons PSAM Ion Pore Domain Ligand(s) ... Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...hM1Dq hM5Dq Human M3 muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al, 2007 rM3D...PMID: 28774929 Guettier JM, Gautam D, Scarselli M, de Azua IR, Li JH, et al (2009). A chemical-genetic ...Forbes SL, Liggett SB (2001). Modification of the beta 2-adrenergic receptor to engineer a receptor-effector...Relationship Studies for Designer Receptors Exclusively Activated by Designer Drugs. ACS Chem Neurosci...

... : An encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9-Blast and lentiGuide-Puro...Off-Spotter: tool for CRISPR/Cas design Program for designing optimal gRNAs. Provides feedback on number of potential...the Chan Zuckerberg Biohub . gRNA Design Deep learning models to design gRNAs and predict their efficiency... a new window) : a webtool that provides models for SpCas9 gRNA design. DeepBE (Link opens in a new window...window) : a webtool that provides models for base editor (BE) gRNA design. DeepPrime (Link opens in a ...window) : a webtool that provides models for prime editor (PE) pegRNA design. PMID 37119812 (Link opens...