...mammalian cells (Cong et al ., Science 2013, Ran et al ., Cell 2013, Ran et al ., Nature Protocols 2013). For ...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,...CRISPR Cas9 for enhanced genome editing specificity. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino...

...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...CASTs is not considered "scarless", as the transposon homology sequences are integrated along with the donor...

...CRISPR plasmids that create double strand breaks in DNA....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...

...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...CRISPR nickase plasmids for the generation of single-strand breaks. CRISPR...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...

...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...binds, Cas9 separates the double stranded DNA target and cleaves both strands near the PAM. (6) The crRNA:...: dCas9 fused to transcriptional activators or repressors can modulate transcription in a reversible manner...palindromic repeat sequences. (1) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA). (2) The...is processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the...:tracrRNA:Cas9 complex unbinds after the double-strand break. CRISPR systems, found in 95% of archaeal...

...repression. The new transactivator rtTA ( r everse t etracycline-controlled t rans a ctivator) was created...TetR On Geley Transactivators (tTA or rtTA) Find a construct that expresses the transactivator for your tetraycline...Overview of tetracycline regulated inducible transcriptional activation or repression Plasmid...tetracycline and not to the TRE, permitting transcription. Tetracycline-dependent promoters are developed... gene expression, a tetracycline-controlled transactivator (tTA) was developed. tTA was created by fusing... of VP16 (virion protein 16), an essential transcriptional activation domain from HSV (herpes simplex ...reduced gene expression. This idea of a hybrid transactivator was initially used with the lac system. Tetracycline...

..., 1163–1171. PMID: 23979020 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D...38688943 Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine..., 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...may be a single-stranded oligonucleotide, double-stranded linear DNA, or a double-stranded DNA plasmid. ...including transposons, integrases, and recombinases, with Cas enzymes. CRISPR Transposases Transposon systems...

...engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...another transfection method such as cationic liposome-based transfection. Optimize transfection conditions... (Crick) strand while sgRNA-B’s protospacer sequence and PAM fall on the top (Watson) strand ( Figure ..., which is used for MEL cells. However, transient transfection of CRISPR/Cas9 plasmids may be successfully...single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the...leading to nonsense mediated decay of the mRNA transcript ( Figure 1B ). Use the example guides for the...protospacer sequence/PAM relative to the top or bottom strand. Determine the reverse complement of each guide...

...- CHGA chromogranin A (parathyroid secretory protein 1) CGA CHGB chromogranin B (secretogranin 1) SCG1...superfamily, member 11 CD254, ODF, OPGL, OPTB2, RANKL, TRANCE, hRANKL2, sOdf TNFSF12 tumor necrosis factor (ligand... perforin and granzyme B, two enzymes that create pores within an infected cell membrane, triggering cell...nuclear transcription factor Y, alpha CBF-A, CBF-B, FLJ11236, HAP2, NF-YA NFYB nuclear transcription factor...RAET1G retinoic acid early transcript 1G ULBP5 RAET1L retinoic acid early transcript 1L - RELB v-rel reticuloendotheliosis...(sodium/bile acid cotransporter family), member 2 ASBT, ISBT, NTCP2 TAP1 transporter 1, ATP-binding cassette...

...for glutamatergic neurotransmission Targeted sensors for glutamatergic neurotransmission. eLife. 2023 Jan...trafficking to the plasma membrane Acute disruption of the synaptic vesicle membrane protein synaptotagmin...measurements of membrane charges A FRET sensor enables quantitative measurements of membrane charges in live...847-56. David Raible pH Red or Orange pH biosensors (pHuji and pHoran4) pHuji, a pH-sensitive red fluorescent...Proteins: Biosensors Metal Ions Small Molecules Neurotransmitters Cell Physiology Browse All You may also like...fluorescent protein (cpFP) or Förster resonance energy transfer (FRET) efficiency of a pair of fluorescent proteins...Calcium Calcium sensor in astrocytes (GCaMP6f), membrane-tethered Khakh Lab pZac2.1 gfaABC1D-lck-GCaMP6f...

... Monomer pET mOrange LIC cloning vector (u-mOrange) - Bacterial Expression pCS2+8CmOrange - Zebrafish/...pCS2+8NmOrange - Zebrafish/Xenopus/C.elegans/Sea urchin mOrange-N1 - Mammalian Expression mOrange-C1 -...Expression mOrange-pBAD - Bacterial Expression mOrange2 549 565 35 6.5 4.5 hr Monomer mOrange2-N1 - Mammalian...Expression LSSmOrange 437 572 23 5.7 2.3 hr Monomer pLSSmOrange-C1 - Mammalian Expression pLSSmOrange-N1 - ...Structure Plasmids mKusabira-Orange (mKO) 548 559 31 5 4.5 hr Monomer pQC mKorange IX - Mammalian Expression...Expression mKusabira Orange-C1 (mKO) - Mammalian Expression mKO-pBAD - Bacterial Expression mOrange 548 562 49 ...Mammalian Expression mOrange2-C1 - Mammalian Expression mOrange2-pBAD - Bacterial Expression mKO2 551 565...

...pOpen-EcoRIM M.EcoRI DNA methyltransferase 165517 pOpen-Eco31IA Eco31IA DNA methyltransferase 165518 pOpen-NotIM...pOpen-NotIM M.NotI DNA methyltransferase 165527 pOpen-Eco31IB Eco31IB DNA methyltransferase 165536 pOpen-PstIM...pOpen-PstIM M.PstI DNA methyltransferase 165538 pOpen-SfiIM M.SfiI 165538 DNA methyltransferase 165547 pOpen-SapIM1...pOpen-SapIM1 M1.SapI DNA methyltransferase 165557 pOpen-SapIM2 M2.SapI DNA methyltransferase 165558 pOpen-EcoRVM...pOpen-EcoRVM M.EcoRV DNA methyltransferase 165577 pOpen-XbaIM M.XbaI DNA methyltransferase 165578 pOpen-NcoIM ...32 single-stranded DNA-binding protein 165549 pOpen-ETSSB Extreme Thermostable Single-Stranded DNA-Binding...Polymerases Restriction Endonucleases Reverse Transcriptases RNA Polymerases Other Enzymes (Link opens in...

...37351 pQC membrane TdTomato IX Membrane Palmitoylation TdTomato Connie Cepko 22479 FUmGW Membrane Palmitoylation...Mitochondria-Outer Membrane Mff acGFP1 Gia Voeltz 158007 pCMV-mGold-Mito-N-7 Mitochondria Cox8A mGold Francois St-Pierre...pCAG-mGFP Membrane Palmitoylation sequence from GAP43 EGFP Connie Cepko 61099 Lck-GFP Membrane Palmitoylation...Mitochondria-Outer Membrane TOMM20 mCherry* Michael Davidson 118736 pBOB-CARMIL2 BH domain-GFP Plasma Membrane CARMIL2...alpha-tubulin mGold Francois St-Pierre 158009 pCMV-mGold-Actin-C-18 Actin Filaments Actin mGold Francois St-Pierre...EGFP Carol Otey 31923 pPaxillin-PSmOrange Focal Adhesions paxillin PSmOrange Vladislav Verkhusha 79999 pZyxin-miRFP703... Proteins are first translated at the endoplasmic reticulum, then are transported to the Golgi apparatus...

...dynamic range, and resistance to photobleaching has made luciferase a common choice in assays ranging from... luciferase. Transcriptional Element Screening Optimized STARR-seq ( S elf- T ranscribing A ctive R egulatory...including transcriptional reporters for NF-kb, TGF-b, c-Myc, p53 and MAPK/JNK transcriptional reporters...collection of luciferase plasmids for assays of transcriptional regulation and bioluminescent reporters. Plasmid...bioluminescence found in a diverse number of organisms, ranging from bacteria to aquatic animals to insects. Luciferase... protein half-lives, making them useful as transcriptional reporters. However, they are not secreted, ...regulatory elements, such as promoters, enhancers and untranslated regions, or the effect of mutations of these...

...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...types of viruses used in research. Lentivirus can transduce both dividing and non-dividing cells without a...stably into the host genome, enabling long term transgene expression. There are some safety considerations...procedures. Additionally, because this virus integrates randomly into the host genome, it has the potential for...encodes the viral genome (typically called the transfer plasmid ). This table lists common lentiviral ...

...enhanced transduction of the retina following subretinal administration and enhanced transduction and spread...cite Boye, et al. 2016. Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-... from the University of Florida for enhanced transduction of the eye and other tissues. As part of our...Y444F) serotype is demonstrated to have enhanced transduction of the retina following intravitreal injection... AAV2(Y444F) serotype also displays enhanced transduction of the trabecular meshwork following intracameral...trpYF) The AAV2(trpYF) serotype shows enhanced transduction of the retina following intravitreal injection...macaque. This serotype also demonstrates enhanced transduction of cells in culture for ex-vivo conditioning...

...Gileadi lab Xenopus SP6 for transcription and injection pCS2P+ - Transcription from the SP6... elegans Transgenic Expression in organisms pBigT - For making transgenic mice. Contains...Tools for nematode transgenesis Rosenberg Lab Plasmids - Transgenic single-copy gene ...expression and packaging Reporters, shRNA expression, transgenics and genome modification Species-Specific...Mammalian CMV, SV40, EF1a, CAG pSG5L Flag HA - Transient expression pWPXL - Lentiviral ...expression pBI-MCS-EGFP - Tet-inducible Transient expression - Gradia Lab plasmids... high expression of UAS-driven transgenes Worm unc-54, variety of worm gene promoters C...

...reprogramming factors Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells...Reprogramming Plasmids Differentiation and Transdifferentiation Plasmids Stem Cell Research Plasmids Stem...differentiated somatic cell into another via transdifferentiation. This method of cell lineage conversion ...technology. Browse Addgene's collection of Transdifferentiation Plasmids below. Many of the techniques for... for reprogramming, differentiation, and transdifferentiation utilize plasmids to increase expression ...human Klf4, Oct4, c-Myc, and Sox2 as VP-16 transcriptional activating fusions in single lentiviral vectors...human iPSC generation OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts...

...For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may...mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA can be present...present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene ...screens using CRISPR AAV transduction CRISPR elements are inserted into an AAV transfer vector and used to ...ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression. The table below summarizes the...details, see Plasmids 101: Mammalian Vectors ) Transient or stable expression of Cas9 and/or gRNA in a ... mammalian cell lines Useful for difficult to transfect cell types, can also be used in vivo A common ...

...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...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined... Edit Cas9 H840A nickase fused to a reverse transcriptase (RT) is capable of installing targeted insertions...