...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...

...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...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 ... 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...

...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...

...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 [...

...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...

...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...

...gRNA-hIRF-1 #12 Expresses a guide RNA (gRNA) to target human IRF-1 promoter 61080 gRNA-hIRF-1 #12/pSIR ..._dCas9-2xAM_hIRF-1 Lentiviral plasmid expressing dCas9-2xAM and gRNA for human IRF-1 promoter. 98041 Sa-dCas9...interest. 98134 hIRF-1 gRNA #409 (Sa) Expresses a guide RNA (gRNA) to target human IRF-1 promoter for the ...system. 105283 hIRF-1 gRNA #351 (Sa) Expresses a guide RNA (gRNA) to target human IRF-1 promoter for the ...Notes. 2018 Feb 27;11(1):154. doi: 10.1186/s13104-018-3262-4. PubMed . Transgenic mouse lines expressing...LexA are subjected to affinity purification (Fig. 1). In enChIP, specific genomic regions are tagged with...plasmids or browse the Fujii Lab web page . Figure 1: iChIP general scheme Figure 2: enChIP general scheme...

...for pMKO.1 puro; Contains shRNA against GFP William Hahn 10676 pMKO.1 GFP Derivative of pMKO.1 puro with...pLKO.1 ‐ TRC control Negative control vector containing non-hairpin insert David Root 1864 pLKO.1 ‐ scrambled...Description PI 8452 pMKO.1 puro Retroviral vector for shRNA expression Bob Weinberg 10675 pMKO.1 puro GFP shRNA ...shRNA expression ID Plasmid Description PI 10878 pLKO.1 ‐ TRC cloning vector shRNA expression; Recommended...protocol for using the popular cloning vector pLKO.1. Trono Lab Lentivectors Toolbox Tyler Jacks Lab Protocols...role for Nramp1 in modifying susceptibility to type 1 diabetes. Kissler S, Stern P, Takahashi K, Hunter ...interfering RNA) via transfection, or shRNA (short hairpin RNA) via transfection or viral delivery of ...

...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...

...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...

...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...the calmodulin in GCaMP. Nat Commun. 2018 Apr 17;9(1):1504. Xiaodong Liu Calcium GCaMP5 genetically encoded...

...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...