...mammalian cells (Cong et al ., Science 2013, Ran et al ., Cell 2013, Ran et al ., Nature Protocols 2013). For ...Heidenreich 1, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F. Nat Biotechnol . 2015 Jan;33(1):102-6...codon-optimized SpCas9; sgRNA can be co-transfected as PCR amplicons (Ran et al., Nature Protocols 2013). Individual...sgRNA can be co-transfected as PCR amplicons using a protocol similar to SpCas9 sgRNA PCR (Ran et al., Nature...and its single guide RNA (sgRNA) into one plasmid (Ran et al., Nature 2015). The dual vector system uses...Genome Engineering using CRISPR/Cas Systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu... Cas9 nucleases. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X,...

...oligos 1:10 in ddH 2 O ( e.g., 1.0 μl annealed oligos + 9.0 μl ddH 2 O to yield a concentration of 1 μM)....35 cycles of (95 °C for 30 sec, 60 °C for 1 min, 72 °C for 1 min), and 72 °C for 10 min. Optimize PCR ...streptomycin and 1% L-glutamine, which is used for MEL cells. However, transient transfection of CRISPR/Cas9...one reaction (see step 3.2 ). Transform 10 μl of DH5α E. coli cells with 1 μl of reaction (from 3.2.1 -...attempting genome editing. Immediately transfer solution from cuvette into 1 ml of culture media after electroporation...engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...sgRNAs manually or using freely available online tools 1 . Use these tools to help identify guide sequences...

...IGHDY1 IGHD1-1 immunoglobulin heavy diversity 1-1 IGHD11 IGHD1-14 immunoglobulin heavy diversity 1-14 (non-...IGHV6-1 immunoglobulin heavy variable 6-1 IGHV61, VH IGHV7-4-1 immunoglobulin heavy variable 7-4-1 IGHV7...TAPBPR THBS1 thrombospondin 1 THBS, THBS-1, TSP, TSP-1, TSP1 TRPC4AP transient receptor potential cation...variable 2-8 IGLV28, V1-2 IGLV3-1 immunoglobulin lambda variable 3-1 IGLV31, V2-1 IGLV3-10 immunoglobulin lambda...NCC-1, NCC1, SCYA13, SCYL1 CCL14 chemokine (C-C motif) ligand 14 CC-1, CC-3, CKb1, FLJ16015, HCC-1, HCC... 3 G0S19-1, LD78ALPHA, MIP-1-alpha, MIP1A, SCYA3 CCL3L1 chemokine (C-C motif) ligand 3-like 1 464.2, D17S1718...GPD, GpFy, WBCQ1 DEFA1 defensin, alpha 1 DEF1, DEFA2, HNP-1, HP-1, MGC138393, MRS DEFA3 defensin, alpha...

...Function PI AV-1-49531P 100040-AAV1 pAAV.hSyn1.Twitch2B.WPRE.SV40 Biosensor Oliver Griesbeck AV-1-50942 50942...Tobias Rose AV-1-PV2723 98929-AAV1 pAAV.hSyn.iGluSnFr.WPRE.SV40 Biosensor Loren Looger AV-1-PV2724 98931... Looger AV-1-PV2725 98932-AAV1 pAAV.CAG.Flex.iGluSnFr.WPRE.SV40 Biosensor Loren Looger AV-1-PV2816 100835... Kim AV-1-PV2817 100839-AAV1 pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 Biosensor GENIE, Douglas Kim AV-1-PV2818 ... Kim AV-1-PV2819 100833-AAV1 pAAV.Syn.Flex.GCaMP6f.WPRE.SV40 Biosensor GENIE, Douglas Kim AV-1-PV2820 ... Kim AV-1-PV2821 100845-AAV1 pAAV.Syn.Flex.GCaMP6s.WPRE.SV40 Biosensor GENIE, Douglas Kim AV-1-PV2822 ...Douglas Kim AV-1-PV2823 100841-AAV1 pAAV.Syn.GCaMP6m.WPRE.SV40 Biosensor GENIE, Douglas Kim AV-1-PV2824 100843...

...none Constitutive 1 Looger 137955 pAAV.CAG.iAChSnFR CAG iAChSnFR none Constitutive 1, 9 Looger 121922 ...Constitutive 1, 9, rg* GENIE 162379 pGP-AAV-syn-FLEX-jGCaMP8f-WPRE Syn jGCaMP8f none Cre dependent 1, 5, 9, ...dependent 1, 5, 9, rg* GENIE 169258 AAV-hSyn-Soma-jGCaMP8f Syn soma-jGCaMP8f none Constitutive 1, 9, rg*...Constitutive 1, 5, rg* Looger 176753 AAV-mDlx-jGCaMP8f-WPRE Dlx jGCaMP8f none Constitutive 1, 9, rg* Looger...Constitutive 1, 9, rg* Looger 176759 pZac2.1-GfaABC1D-lck-jGCaMP8f GfaABC1D jGCaMP8f none Constitutive 1, 9 Looger...Constitutive 1, 5, 9, rg* GENIE 162377 pGP-AAV-syn-FLEX-jGCaMP8s-WPRE Syn jGCaMP8s none Cre dependent 1, 9, rg...dependent 1, 9, rg* GENIE 167572 AAV-hSyn-Ribo-jGCaMP8s Syn ribo-jGCaMP8s none Constitutive 1 Fyhn 169256...

...Constitutive 1, 2, 5, 8, 9, rg*, PHP.eB, PHP.S Boyden 83900 pAAV-mDlx-GFP-Fishell-1 Dlx GFP Constitutive 1, 2, ...Constitutive 1, 2, 5, 8, 9, rg*, PHP.eB Roth 50469 pAAV-CaMKIIa-EGFP CaMKIIa EGFP Constitutive 1, 2, 5, 8,...Constitutive 1, 2, 5, 8, 9, rg*, PHP.eB Deisseroth 114470 pAAV-Ef1a-mCherry EF1a mCherry Constitutive 1, 2, 5...Constitutive 1, 8 Deisseroth 135630 pAAV-S5E2-dTom-nlsdTom E2 regulatory dTomato Constitutive 1, 9, PHP.eB... dependent 1, 2, 5, 9, rg* Deisseroth 28306 pAAV-FLEX-tdTomato CAG tdTomato Cre dependent 1, 2, 5, 8, ...dependent 1, 2, 5, 8, 9, rg* Harvey 131002 pAAV-Ef1a-DIO mScarlet EF1a mScarlet Cre dependent 1, 5 Deisseroth...Cre dependent 1, 2, 5, 8, rg* Roth 50459 pAAV-hSyn-DIO-mCherry hSyn mCherry Cre dependent 1, 2, 5, 8, 9,...

...-phase expressed 1; also known as GTSE1 BAI-1 Brain-specific angiogenesis inhibitor 1 Bax BCL2-associated...inhibitor type 1), member 1 PERP TP53 apoptosis effector PIDD p53-induced death domain protein 1 PIGs Etoposide...receptor superfamily, member 10b E2F-1 E2F transcription factor 1 Fas Fas cell surface death receptor ... Name 14-3-3-σ Stratifin Apaf-1 Apoptotic peptidase activating factor 1 ATM ATM serine/threonine kinase...kinase 4 or 6 CHK1 Checkpoint kinase 1 CHK2 Checkpoint kinase 2 Cop-1 Ring finger and WD repeat domain 2...SESN1 SESN2 SESN3 Sestrins 1, 2, or 3 Siah Siah E3 ubiquitin protein ligase 1 TSC2 Tuberous sclerosis 2...Noxa Phorbol-12-myristate-13-acetate-induced protein 1; also known as PMAIP1 p14ARF Cyclin-dependent kinase...

...Cre.SV40 CamKII none 1, 5, 9, rg* Wilson 105537 pENN.AAV.CMVs.Pl.Cre.rBG CMV none 1, 2, 5, 8, 9, rh10 Wilson...-Cre EF1a none 1, 5, 8, 9, rg* Engel , Nectow 69570 pAAV-EF1a-N-CretrcintG EF1a none 1 Cepko 69571 pAAV-EF1a-C-CreintG...pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40 Syn eGFP 1, 2, 5, 8, 9, rg*, PHPeB Wilson 105553 pENN.AAV.hSyn.Cre.WPRE.hGH Syn none 1, 2, 5, 8, 9,...AAV-hSyn-mCherry-P2A-Cre-WPRE Syn mCherry 1 Yang 107738 pAAV-hSyn-Cre-P2A-dTomato Syn dTomato 1, 2, 5, 8, 9, rg*, PHPeB ...mCherry (not a fusion tag) 1, rg* Deisseroth 55637 pAAV-EF1a-Flpo EF1a none 1, 2, rg* Deisseroth 87306 ...pEF1a-DIO-FLPo-WPRE-hGHpA EF1a none 1, 2, 5, 8, 9, rg* Zhang 75469 pAAV-EF1a-Flp-DOG-NW EF1a none 1 Cepko 183412 pAAV-CAG-FlpO... pAAV-EF1a-iCreV EF1a none 1, PHPeB Zeng 140136 pAAV-EF1a-iDreV EF1a none 1, PHPeB Zeng 140137 pAAV-EF1a-iFlpV...

..., hRev, THRA1, THRAL, ear-1 nuclear receptor subfamily 1, group D, member 1 NR1D2 Plasmids NR1D2, RVR,...subfamily 1, group D, member 2 NR1H3 Plasmids NR1H3, LXR-a, LXRA, RLD-1 nuclear receptor subfamily 1, group...AHX, DAX-1, DAX1, DSS, GTD, HHG, NROB1, SRXY2 nuclear receptor subfamily 0, group B, member 1 NR1D1 Plasmids...member 1 NR3C1 Plasmids NR3C1, GCCR, GCR, GR, GRL nuclear receptor subfamily 3, group C, member 1 (glucocorticoid...HMR, N10, NAK-1, NGFIB, NP10, NUR77, TR3 nuclear receptor subfamily 4, group A, member 1 NR4A2 Plasmids... FTZF1, POF7, SF-1, SF1, SPGF8, SRXY3 nuclear receptor subfamily 5, group A, member 1 NR5A2 Plasmids NR5A2...important regions of nuclear receptors are: 1) a ligand binding domain, which interacts with hormones or...

...190293 Anti-AMIGO-1 [L86/14] AMIGO-1 Mouse Mouse IgG2a 190294 Anti-AMIGO-1 [L86/2] AMIGO-1 Mouse Mouse IgG2a...ATPase 1 [L60/4R-2b] Copper ATPase 1 Human Mouse IgG2b 206648 Anti-AMIGO-1 [L86/33R-2b] AMIGO-1 Mouse ...channel [L6/60R-1] Slo1 K+ channel Mouse Mouse IgG1 206695 Anti-AMIGO-1 [L86/33R-1] AMIGO-1 Mouse Mouse IgG2a...Anti-Olig1 [N149/25R-1] Olig1 Mouse Mouse IgG1 206702 Anti-Neurexin-1-Beta [N170A/1R-1] Neurexin-1-Beta Human Mouse...222160 Beclin-1 [N248A/15R] Beclin-1 Mouse Mouse IgG2a 222161 Beclin-1 [N248A/22R] Beclin-1 Mouse Mouse ...Mouse Mouse 190534 Neurexin-1-Beta scFv [N170A/1] N170A/1 scFv Neurexin-1-Beta Human Mouse 190535 PSD-...Mouse 206755 IP3 receptor, type 1 scFv [L24/1] L24/1 scFv IP3 receptor, type 1 Rat Mouse 206756 VSP scFv [...

...Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 PLXNB1 Plexin B1 PPP1CA Protein phosphatase 1 catalytic ... dependent protein kinase 1 PEBP1 Phosphatidylethanolamine binding protein 1 PIK3 Catalytic Subunits PIK3CA...family member RB1 Retinoblastoma 1 RCE1 Ras converting CAAX endopeptidase 1 RHEB Ras homolog enriched in ...protein of MTOR, complex 1 SAV1 Salvador family WW domain containing protein 1 SCRIB Scribbled planar cell...guanine nucleotide exchange factor 1 YAP1 Yes associated protein 1 Return to top Resources RAS Pathway...kinase 11 TBK1 TANK binding kinase 1 TFDP TFDP1 TFDP2 Transcription factor Dp TIAM TIAM1 TIAM2 T-cell ...Mechanistic target of rapamycin NF1 Neurofibromin 1 NFE2L2 Nuclear factor, erythroid 2 like 2 NFKB1 Nuclear...

...Constitutive 1, 9 Svoboda 83898 pAAV-mDlx-ChR2-mCherry-Fishell-3 Dlx ChR2 mCherry Constitutive 1, 9, rg* Fishell...Constitutive 1, 2, 5, 9 Deisseroth 26973 pAAV-hSyn-hChR2(H134R)-EYFP Syn ChR2/H134R EYFP Constitutive 1, 2, 5...Constitutive 1, 5, rg* Boyden 59171 pAAV-Syn-ChrimsonR-tdT Syn ChrimsonR tdTomato Constitutive 1, 5, 9 Boyden...dependent 1, 5, 9, rg* Deisseroth 26971 pAAV-CaMKIIa-eNpHR 3.0-EYFP CaMKII eNpHR 3.0 EYFP Constitutive 1, 9 ...Constitutive 1, 5, rg* Yizhar 125713 AAV-hSyn1-SIO-eOPN3-mScarlet-WPRE Syn eOPN3 mScarlet Cre dependent 1, 5 Yizhar...Constitutive 1, 5 Yizhar 198511 pAAV_hSyn-DIO-PdCO-mScarlet-WPRE Syn PdCO mScarlet Cre dependent 1, 5 Yizhar...Constitutive 1, 5 Yizhar 198516 pAAV_EF1a-DIO-PdCO-mScarlet-ER-miniWPRE EF1a PdCO mScarlet Cre dependent 1, 5 Yizhar...

... cleaves it to destroy the invader ( Figure 1 ). Figure 1: An overview of the endogenous Type II bacterial...short palindromic repeat sequences. (1) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA...Nature . 550(7676):407-410. PMID: 28931002 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...CRISPR-Cas9. Cell . 167(7):1829-1838. PMID: 27984730 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...engineering, with Type V following in 2015. Class 1 (Multi-subunit effector complex) Class 2 (Single multi-domain...enChIP) using CRISPR. Biochem Biophys Res Commun . 439(1):132-6. PMID: 23942116 Gilbert LA, Horlbeck MA, Adamson...CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol . 35(1):31-34. PMID: 27918548...

...none 1, 9 Roth 83896 pAAV-hDlx-GiDREADD-dTomato-Fishell-5 hM4D(Gi) - Inhibition NLS-dTomato none 1, 9, ...hM3D(Gq) - Activation mCherry fusion Cre-dependent 1, 2, 5, 8, 9, rg*, PHPeB Roth 44362 pAAV-hSyn-DIO-hM4D...hM4D(Gi) - Inhibition mCherry fusion Cre-dependent 1, 2, 5, 8, 9, rg*, PHPeB Roth 65417 pAAV-hSyn-dF-HA-KORD-IRES-mCitrine...mCherry hM3D(Gq) - Activation mCherry fusion none 1, 2, 5, 8, 9, rg* Roth 50475 pAAV-hSyn-hM4D(Gi)-mCherry...mCherry hM4D(Gi) - Inhibition mCherry fusion none 1, 2, 5, 8, 9, rg* Roth 52536 rAAV-CAG::FLEX-rev:: hM4D...hM4D-2a-GFP hM4D(Gi) - Inhibition GFP Cre-dependent 1 Sternson 154867 pAAV-hSyn-fDIO-hM4D(Gi)-mCherry-WPREpA...mCherry hM3D(Gq) - Activation mCherry fusion none 1, 2, 5, 8, 9 Roth 50477 pAAV-CaMKIIa-hM4D(Gi)-mCherry...

...some mammalian systems have been developed. Figure 1: Overview of the basic CAST mechanism. Created with...CRISPR-associated transposases (CASTs) combine the large-scale capacity of transposases (Tns) with the precise...Find plasmids for CRISPR-associated transposases (CASTs) for insertion of donor DNA. CRISPR...CRISPR Plasmids: CRISPR Transposases (CASTs) Browse CRISPR Plasmids By Function Genome Editing Cut Base...Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...lacks nuclease activity, instead recruiting the transposase (comprised of multiple components) to target ...locations in the genome defined by a guide RNA. The transposase can then integrate donor DNA. Editing by CASTs...

...accumulation in bacteria. Biotechnol Biofuels. 2008 Jun 3. 1(1):11. Wolf Frommer Maltose Green fluorescent MBP-based...the plasma membrane Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff...cassette of green- and orange-emitting fluorescent proteins. Nat Commun. 2017 Sep 5;8(1):431. Wolf Frommer...Sorting On Transient Cellular Responses Of Genetically-Encoded Sensors. Anal Chem. 2017 Jan 3;89(1):711-719...physiology. Nat Commun. 2020 Aug 4;11(1):3881. Adam Cohen Chloride (Cl-) Membrane-targeted biosensor for monitoring...of cellular physiology. Nat Commun. 2020 Aug 4;11(1):3881. Adam Cohen Calcium GCaMP6f expression in forebrain...Dynamics in High-Ca Organelles. Cell Chem Biol. 2016 Jun 1. pii: S2451-9456(16)30163-5. Teresa Alonso , Javier...

... Human Doench 3rd 1, 2, or 4 49,766 arrays Broad GPP kinome Brunello 75314, 75315 (1 plasmid) 75312, 75313...Version 1 1000000069 Knockout Human Moffat 3rd 12 176,500 Toronto KnockOut - Version 3 90294 (1 plasmid...11,108 Borodina Yeast Transporter Libraries 153101-153106 Knockout Yeast Borodina N/A 1 Varies Bovine CRISPR...pgRNAs 1,718 Broad GPP genome-wide Brunello 73179 (1 plasmid) 73178 (2 plasmid) Knockout Human Doench and...Root 3rd 4 76,441 Broad GPP genome-wide Brie 73632 (1 plasmid) 73633 (2 plasmid) Knockout Mouse Doench and...and Root 3rd 4 3,052 Broad GPP kinome Brie 75317 (1 plasmid) 75316 (2 plasmid) Knockout Mouse Doench and...Human Birsoy 3rd 8 20,051 Human GeCKO v2 1000000048 (1 plasmid) 1000000049 (2 plasmid) Knockout Human Zhang...

...rapamycin NF1 Neurofibromin 1 PRAS40 Also known as AKT1S1; AKT1 substrate 1 (proline rich) PTEN Phosphatase...Symbol Name 4E-BP Eukaryotic translation initiation factor 4E, binding protein 1 Cyclin D1 Also known as CCND1...RPTOR; Regulatory associated protein of MTOR, complex 1 Rheb Ras homolog enriched in brain S6K Also known ...Mitogen-activated protein kinase associated protein 1 mTOR Mechanistic target of rapamycin p53 TP53; tumor...protein p53 PDK1 Pyruvate dehydrogenase kinase, isozyme 1 PI3K Catalytic Subunits PIK3CA PIK3CB PIK3CD PIK3CG...complex 2 SGK Serum/glucocorticoid regulated kinase 1 Return to top Resources Cancer Pathway ORF Kit (Sabatini...Loss of the tumor suppressor p53 also promotes aberrant mTORC1 activity, and multiple familial cancers...

...Typical Titer Small 10 × 100 µL aliquots 1 mL 4 × 10 12 GC/mL* 1 × 10 13 GC/mL* Medium 25 × 100 µL aliquots...serotype (AAV1, AAV2, AAV5, AAV8, or AAVrg) and volume (1 mL, 2.5 mL, or 5 mL). Currently, we can only package...to 10 weeks. This breaks down as follows: Request 1–2 days Look for the banner on an eligible plasmid ...place your request. We will send you an email within 1-2 days with a response. Most requests will be approved... we can begin processing your order. MTA Approval 1+ business days, varies by requesting organization ...scientist through an MTA. This process typically takes 1–4 days, depending on your institution. AAV Production...tools that effectively transduce many mammalian cell types. AAV is used in a wide range of experiments, both...

...the tet-off transactivator (tTA) with a positive feedback loop for amplified tTA expression. 1 Gradinaru...expression of membrane-bound GFP and synaptophysin-mRuby for labeling of axon terminals. 1 Luo 60658 pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato...Tetracycline-inducible Cre-dependent, Tet-inducible expression of EYFP 1 Gradinaru Tools for Affinity Purification These AAV...synaptophysin-EGFP for labeling of axon terminals. 1 Zeng 71760 pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby...from Addgene's viral service encoding tet-off transactivators and tools for affinity purification (TRAP)....Packaging Service: Molecular Tools Tetracycline Transactivators Affinity Purification Neurophysiology Cell ...

...researchers use ssDNA or dsDNA repair templates with 1. homology to the DNA flanking the DSB and 2. a specific...CRISPR plasmids that create double strand breaks in DNA. CRISPR...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...

...alpha-actinin 1 Focal Adhesions alpha-actinin 1 EGFP Carol Otey 31923 pPaxillin-PSmOrange Focal Adhesions...Brown 11908 pEGFP-N1 alpha-actinin 1 Actin Filaments alpha-actinin 1 EGFP Carol Otey 27676 Cortactin-pmCherryC1...Autophagosome Sequestosome-1 EGFP Noboru Mizushima 38272 pMXs-IP GFP-WIPI-1 Autophagosome WIPI1 EGFP Noboru...apparatus eNOS(1-33) CFP Alexandra Newton 36205 pmTurquoise2-Golgi Golgi apparatus B4GALT1(1-61) mTurquoise2...Shirihai 158003 pCMV-mGold-CAF1-C-10 Nucleus CAF-1 mGold Francois St-Pierre 27705 CAV1-mCherry Caveolae Cav1...COXIV-COX8-dL5-2XG4S-mCer3 Mitochondria COX IV-derived (1-22 aa) import sequence and COX VIII signal peptide...Verkhusha 36208 pmTurquoise2-Mito Mitochondria COX8A(1-29) mTurquoise2 Dorus Gadella 26673 pEGFP-C1-Fibrillarin...

...Cre-GFP fusion EF-1 alpha Mammalian Sauer 11923 pBS598 EF1alpha-EGFPcre Cre-EGFP fusion EF-1 alpha Mammalian...mCherry coexpression EF-1 alpha AAV Deisseroth 55635 pAAV-EF1a-sCre SCre EF-1 alpha AAV Deisseroth 55636...55636 pAAV-EF1a-Cre Cre EF-1 alpha AAV Deisseroth 55638 pAAV-EF1a-vCre VCre EF-1 alpha AAV Deisseroth 59701...) EF-1 alpha AAV Cepko 69571 pAAV-EF1a-C-CreintG Cre recombinase dependent on GFP (CRE-DOG) EF-1 alpha...deletion, and translocation. Figure 1. Recombination outcomes are determined by target site orientation... Mammalian Sauer 11918 pBS513 EF1alpha-cre Cre EF-1 alpha Mammalian Sauer 11919 pBS448 RSV-GFPcre Cre-...11955 pBS505 EF1alpha-EGFPcre* Cre-EGFP fusion EF-1 alpha Mammalian Sauer 11956 pBS594 promoterless EGFPcre...

...AdEasier®-1 cells (strain) - Bacterial strain that contains AdEasy®-1 plasmid... vector for transgene expression under a CMV promoter pAdEasy®-1 - Recombine ...Tobacco mosaic virus (TMV)-based transient expression vector GFP Varies pMKO.1 GFP - Retroviral ...N-terminal Myc tag for mammalian expression pGEX-4T-1-3xMyc - Bacterial vector for Myc tag pETcon(-) - Yeast...PGKpuro2ABFP - For retroviral delivery of one sgRNA pMKO.1 GFP - Retroviral shRNA expression Retroviral backbones... Tet-inducible lentiviral shRNA expression pLKO.1 - TRC cloning vector - Lentiviral shRNA expression...driven puromycin Hygromycin Mammalian, Varies pLKO.1 hygro - Lentiviral shRNA expression lentiCRISPRv2 ...

...Fawzi 127194 RP1B FUS 1-163 QQ4xSS #1 FUS His T7 ALS Nicolas Fawzi 127195 RP1B FUS 1-163 QQ4xSS #2 FUS His... type 1 scFv [L24/1] ITPR1 CMV Spinocerebellar ataxia James Trimmer 206790 IP3 receptor, type 1 scFv [...Cookson 13321 pGEX5X.1 PINK1 WT PINK1 GST tac Parkinson's Mark Cookson 13322 pGEX5X.1 PINK1 KD PINK1 GST... His, V5 EF-1 alpha Parkinson's Mark Cookson 25081 pDEST51-LRRK2-R1441C LRRK2 His, V5 EF-1 alpha Parkinson's... His, V5 EF-1 alpha Parkinson's Mark Cookson 25083 pDEST51-LRRK2-R1441H LRRK2 His, V5 EF-1 alpha Parkinson's...Cookson 29350 pGEX-5X-1-DJ1-WT PARK7 GST tac Parkinson's Mark Cookson 29351 pGEX-5X-1-DJ1-D149A PARK7 GST...Cookson 29352 pGEX-5X-1-DJ1-E64D PARK7 GST tac Parkinson's Mark Cookson 29353 pGEX-5X-1-DJ1-M26I PARK7 GST...

...mEGFP Sialyltransferase 1 Golgi 109122 NPM1-mEGFP AICSDP-50 mEGFP Nucleophosmin Nucleolus (granular component...Institute ID Tag Protein Structure 87420 PXN-EGFP AICSDP-1 EGFP Paxillin Matrix Adhesions 87421 TUBA1B-mEGFP ...TJP1-mEGFP AICSDP-23 mEGFP Tight junction protein ZO-1 Tight junctions 91565 AAVS1-mEGFP AICSDP-35 mEGFP ...Centrioles 101782 LAMP1-mEGFP AICSDP-19 mEGFP LAMP-1 Lysosome 101783 MAP1LC3B-mEGFP AICSDP-25 mEGFP Autophagy-related... HIST1H2BJ-mEGFP AICSDP-52 mEGFP Histone H2B type 1-J Histones 109119 CTNNB1-mEGFP AICSDP-47 mEGFP Beta-catenin...124606 SOX2-mEGFP AICSDP-60 mEGFP Transcription factor SOX-2 Transcription Factor 124607 ACTN2-mEGFP AICSDP...Nucleolus (granular component) 133963 UBTF-HaloTag AICSDP-80 HaloTag Nucleolar transcription factor UBF...

...pCDH-EF1-DIO-copGFP 72253 Expresses copGFP under EF-1 promoter when Cre is expressed by another vector pCDH-EF1...pCDH-EF1-DIO-tdTomato 72254 Expresses tdTomato under EF-1 promoter when Cre is expressed by another vector pCDH-CB-iCre-P2A-tdTomato-T2A-Puro...vector pCDH-EF1-FLPe 72262 Expresses FLPe from the EF-1 promoter pCDH-EF1-copGFP-T2A-Puro 72263 Express copGFP...pCDH-EF1 72266 Express gene of interest from the EF-1 promoter pCDH-CB 72267 Express gene of interest from...72484 Express gene of interest from a truncated EF-1 promoter pCDH-EF1-Luc2-P2A-copGFP 72485 Expresses ...lentiviral and adenoviral transfer vectors with Addgene. Lentiviral Transfer Vectors Within this set of...packaging these transfer vectors and generating infectious virus. Protocol: Lentivirus Transfer Vector Production...

...plasmid Vogelstein 16399 AdEasier-1 BJ5183 derivative that contains AdEasy-1™ plasmid Vogelstein Adenoviral...adenoviral transfer plasmids that have inserts. ID Plasmid Type Description PI 16400 pAdEasy-1 Adenoviral...for the AdenoBuilder genome assembly system. Block 1 with deletion in E1 and insertion of GFP expression...levels of transgene expression. Additionally, these viruses are able to target a broad range of dividing...genome. Disadvantages of adenoviruses include transient transgene expression, as well as the substantial immune...a class of medium-sized, non-enveloped, double-stranded DNA viruses known to cause mild to severe respiratory...both plasmids. This DNA molecule can then be transfected into mammalian packaging cell lines to generate...

...library as GST-fusion peptides (“mode #1” expression). The mode #1 phosphosite library ( 111704 ) is available...standards for quantitative assessments. The mode #1 phosphosite library can be generated with either phosphoserine...available from Addgene. Screening kinases. The mode #1 phosphosite library can also be synthesized using ...pSerOTS-C1* (V70) Pooled Library 188526 iSPI_pSer_Subpool#1 Pooled Library 188527 iSPI_pSer_Subpool#2 Pooled Library...split mCherry 14-3-3β Pooled Library 111704 Mode #1 Library Pooled Library 111705 Mode #2 Library (14-...partial UAG codon reassignment and release factor 1 deletion. Heinemann IU, Rovner AJ, Aerni HR, Rogulina...optimization of an orthogonal translation system with enhanced biological tolerance. Mohler K, Moen JM, Rogulina...

...TATGGTGTCAAACCACAGtgcatgatgactgtctTTCCAGTCTGACCGATGA Dlk1 (site #1) TAL3240 & TAL3241 TGGTGGCGCAGCAGAGGGagctgatccaggaccaGGCCACCGTGAACATCA...TACAAGACAGGGGACAGAagctaaactcatggactgCACAGCTAAGGTAAGA hemogen (site #1) TAL3274 & TAL3275 TGGAAGACCCGTTGGAGAaagagatcccaccaacTGAAATAAAAGATTCAGA...TCTTCCGTTTCCACATCCaccacatcccaacagagcAGCGGGAGCAGCAGTAAA hif1al (site #1) TAL3278 & TAL3279 TGTTTCAGCAGAGCCCCGctgaagagctccccatGGAGATGGAAGGAGTGGA...TGATCCTCTGGCCCATTAacgactcctgggccaaCTCAAGTAGGGGAAACGA Park2 (site #1) TAL 3012 & TAL 3013 TGGAGCAGGGTGCGAGTGtgtctgagctgaaggaGGCGGTGGGTCGTTTACA...TTTCCTCTCATAGTCAATattaattctctatttgGCTCAAAATGTCAGTAAA Plekho1b-1 TAL3142 & TAL3143 TCGTCAAAGCGGGGCCCTcaggatgccaatcaacAGCCTGTGCAGCCCGACA...TATAGCATGATGATGGAAacggaccttcattcccCGGGACCCCAAACCAACA sox2 (site #1) TAL3178 & TAL3179 TGGAAACCGAGCTGAAGCccccggcgccccagcccaACACCGGGGGCACGGGGA...TTGCTCCTTGGTTGCATCtctgtcttcattatcaCAAACATTTTCTTCATTA tetmethylcytosinedioxygenase 1 TAL3572 & TAL3573 TCAACTCGCGCATCCACAaagcgaaatgttaagaGGGTGAAGGCTTCCATGA...

...endogenous repair mechanisms. Cas3 belongs to the Class 1 family of CRISPR systems, the most abundant type found...bacteria and archaea. While abundant in nature, Class 1 systems are largely underutilized compared to their...still separate the individual Cas components. Figure 1: Overview of Cascade-Cas3 mechanism. Created with ...non-targeted strand, Cas3 starts unraveling DNA using its helicase properties, cleaving the targeted strand non-specifically...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...After nicking the target DNA, Cas3 initiates single-strand DNA degradation. This degradation can continue ...non-specifically along the way. Cas3 initiates a final double-strand break, either due to a boundary defined by anti-CRISPR...

...Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann...pooled format into lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9..., 1163–1171. PMID: 23979020 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D..., 17 (5), 471–479. PMID: 32203383 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....CRISPR-Cas9. Cell , 167 (7), 1829-1838.e9. PMID: 27984730 Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott...Nature Protocols , 8 (11), 2281–2308. PMID: 24157548 Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg...Communications , 14 (1), 6175. PMID: 37794046 Small precision edits Anzalone, A. V., Randolph, P. B., Davis,...

...constitutive transgene expression and EGFP coexpression. Trono 17619 EF.CMV.RFP 2nd EF-1 alpha promoter...working with lentivirus: these viruses are based on HIV-1, which may require additional lab biosafety procedures...". ID Plasmid Generation Description PI 8453 pLKO.1 puro 3rd U6-driven shRNA empty plasmid with puro resistance...plasmid 24150 for hygro resistance. Weinberg 10878 pLKO.1 – TRC cloning plasmid 3rd U6-driven shRNA empty plasmid...by plasmids called "transfer plasmids," which can be modified to encode a wide range of gene products. ...Addgene lentiviral packaging, envelope, and transfer plasmids Viral...Libraries All Viral Plasmids Packaging Plasmids Transfer Plasmids Lentiviruses, a subset of retroviruses...

...astrocytes by defined transcription factors. Stem Cell Reports. 2015 Jan 13;4(1):25-36. Broccoli Fibroblasts...Protoc Cell Biol. 2018 Jun;79(1):e51. Next generation vectors for transcription factor mediated differentiation... with CRISPR activators. Nat Commun. 2018 Jul 6;9(1):2643. Otonkoski Lentivirus Human Expression of human...cell reprogramming. Stem Cell Res Ther. 2017 Jun 5;8(1):132. Zovein Replicating EBNA1 episome Human Non-integrating...vector. Proc Natl Acad Sci U S A. 2009 Jan 6. 106(1):157-62. Jaenisch Lentivirus Mouse Doxycycline-inducible...peripheral sensory neurons. Nat Neurosci. 2015 Jan;18(1):25-35. Baldwin Fibroblasts iTSCs Lentiviral Mouse... from Human ALS Patients. Cell Rep. 2016 Jan 5;14(1):115-28. Zhang Adult Dermal Fibroblasts Neurons Lentiviral...

...Barcode Library Version 1 Ready-to-use lentiviral particles carrying version 1 of the CellTag barcoding...barcoding library. Version 1 of the Celltag library contains 19973 barcodes to combinatorially index cells for... used to generate double-stranded breaks ( DSBs or cuts ) or single-stranded breaks ( nicks ). ID Name...Cas9 (dCas9) can be fused to a transactivator and used as a transcriptional activator . ID Name Insert Antibiotic... in a new window) . Please note this does not guarantee viral service, but lets us know what viruses would...

... design and delivery, see resources below. Figure 1: Overview of shRNA-mediated RNA interference. Created...Publication Additional Resources Addgene Resources pLKO.1 cloning vector protocol Other Addgene viral protocols... the double-stranded siRNA and degrades one of the strands. The remaining single-stranded siRNA then binds...silencing is achieved through the delivery of a double-stranded RNA (dsRNA) that matches the mRNA target sequence...short interfering RNA). Delivery can occur via transfection or viral delivery of a plasmid. shRNAs are often...vector is taken up by a host, the viral genome is transcribed, producing the shRNA. Dicer, an RNase III enzyme...

...rabies virus (RABV) for monoclonal neuronal tracing. (1) AAV encoding TVA and G-protein is delivered to the...with a deletion-mutant rabies virus. Nat Methods. 4(1):47-9. PMID: 17179932 Wickersham IR, Lyon DC, Barnard...glycoprotein (G) gene, which is not required for the transcription or replication of the genome within infected...infected cells, but is required for transynnaptic spread (Wickersham et al., 2007a, Wickersham et al., 2007b...essential G protein is provided to the starter cell in trans , via delivery by an AAV. Since this starter cell... cell using the G-protein, which is supplied in trans . (3) Nascent G-protein coated RAVB is released ...Callaway EM. 2007b. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons...

...U6:1_U6:3tandemgRNAs dU6:1 and dU6:3 BbsI Injection or in vitro transcription Virmilion none, need Cas9...Wildonger 49408 pCFD1-dU6:1gRNA dU6:1 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ... BspQI Transfection Puromycin yes, cut Ji-Long Liu 45946 pU6-BbsI-chiRNA dU6 BbsI Transfection none, need...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...

...10676 pMKO.1 GFP MoMLV U6-driven plasmid for shRNA expression; also expresses GFP. See pMKO.1 puro for ...reverse transcriptase and integrase. Once in a target cell, the virus uses the reverse transcriptase in tow...long-term transgene expression. Some disadvantages of γ-retroviruses are that they can only transduce dividing...Kasparov Popular γ-Retroviral Transfer Plasmids Common γ-retroviral transfer plasmids. Want a retroviral...Plasmid for transgene expression; also expresses luciferase Lowe 9044 pMIG MSCV Plasmid for transgene expression...Addgene, including popular packaging, envelope, and transfer plasmids. Viral...envelope). Additionally, γ-retroviruses integrate randomly into the host's genome, which can lead to oncogenesis...

...element Gordon Fishell 83900-AAV1 pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons...element Gordon Fishell 83900-AAV2 pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons...element Gordon Fishell 83900-AAV5 pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons...element Gordon Fishell 83900-AAV8 pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons...element Gordon Fishell 83900-AAV9 pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons... Gordon Fishell 83900-AAVrg pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons... Gordon Fishell 83900-PHPeB pAAV-mDlx-GFP-Fishell-1 GFP expression in forebrain GABA-ergic interneurons...