We narrowed to 84 results for: cas9 expression construct

...Selection for cells stably and highly expressing these constructs can be achieved by fluorescence-activated cell...sorting, and stable gRNA expression can be achieved using lentiviral backbones expressing blasticidin or puromycin...several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection...and gene expression (Sapozhnikov and Szyf 2021).    Figure 3: A simpler approach - dCas9 blocks ...especially 3’ of PAM) might work too.  High dCas9 and gRNA expression are key for complete DNA demethylation...balance these needs is the lentiviral-based expression of floxed dCas9 so that it may later be excised by Cre...could be affected, leading to unpredicted gene expression changes (Sapozhnikov and Szyf 2021, Charlton ...

... have found that expression occurs quickly and results in long and stable expression (up to 9 months in...the onset of gene expression. However, it has yet to be determined what gene expression looks like at 3 ...To decrease the time until expression, it is possible to use AAV constructs with self-complementing structure...Versatile Viral Tool for Gene Expression in Mammals A Match Made in Heaven: CRISPR/Cas9 and AAV Viral Vector Elements...AAV vectors to deliver chemogenetic/optogenetic constructs into our cells of interest. Although they have...AAV2 can transduce many tissue types, it has low expression in several brain regions where other serotypes... under study. Many AAVs will show significant expression in a variety of cell types, but you should try...

...combining specific sgRNAs with Cas9 or Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks...with the sgRNA expression vector allowing the screen to be conducted with only two constructs (the other ... loss of function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or ...sgRNA library In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation... activation domain (CRISPRa) to regulate gene expression. Some CRISPR libraries contain many sgRNAs per... issues. CRISPRi libraries utilize an engineered Cas9, which contains multiple point mutations in the ...screens can help reveal the function of lowly expressed genes or genes that are more drastically affected...

...by tethering cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting ...Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9 Target-AID (PmCDA1...platforms. Hua et al. constructed a number of plant base editors replacing the SpCas9 with the mutant versions...A>G or T>C mutations in the plant genome. We constructed three versions of the vector (pKABE6.3, pKABE7.9...scaffold) in a pol-III (OsU3) promoter driven expression system. We use a single polycistronic tRNA-gRNA...Agrobacterium-mediated transformation. We also constructed smaller vectors suitable for transient assays...Bastian Minkenberg, and Yinong Yang. "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous...

...lentiviral constructs. In 2007 we continued to see requests grow for lentiviral packaging and expression vectors...increase in popularity of empty backbones for shRNA expression, such as pLKO.1 puro and pLKO.1 – TRC cloning...empty backbones such as pBABE-puro for retroviral expression and pLVTHM, a lentiviral plasmid for mammalian...technology of the year in our collection. CRISPR/Cas9 - At Addgene, 2013 will be known as the "Year of.... So far, we have distributed over 10,000 CRISPR/Cas9 plasmids to more than 3,000 labs around the world...

...control expression of their genes of interest. While recombinases are powerful gene expression tools in...inducible Cre for precise control of bacterial gene expression By Meghan Rego Recombinases are enzymes that ...conformational changes in a protein.    Cells expressing an RFP reporter plasmid for recombinase activity... ~70kDa, with a molecular weight roughly half of Cas9 or Cas12a. This is in part due to the fact that ...large heterogeneous cultures.  HeLa cells expressed a fusion of keratin with mGold (top) and mVenus...SPOTlight was used to image over 3 million cell types expressing variants of Yellow Fluorescent Protein (YFP)....CRISPR Plasmids and Resources page.  CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair) is a double-strand...

...lentiviral expression platform, to assess selective degradation of FKBP12F36V-BRD4 using CRISPR/Cas9-mediated...engineered to express ENL-FKBP12F36V and endogenous ENL was inactivated using CRISPR/Cas9 gene-editing....engineered to express FKBP12F36V-MELK and endogenous MELK was inactivated using CRISPR/Cas9 gene-editing... Cell lines were engineered to express YY1-FKBP12F36V using CRISPR/Cas9-mediated knock-in. Featured molecule...molecule FKBP12F36V degraders (dTAG molecule) and expression of FKBP12F36V-tagged proteins. The dTAG molecule...from Addgene. These plasmids enable lentiviral expression of N- or C-terminally FKBP12F36V-tagged fusion...detailed information on cloning procedures used to construct the deposited plasmids. Molecules: We synthesized...

...like Cas9. This is where TnpB offers a clear advantage. At approximately one-third the size of Cas9 or ...nuclease could help keep the total construct size smaller than Cas9 alone. In our recent study, we demonstrated... most well-known CRISPR-associated nucleases are Cas9 and Cas12a, the heavyweights in the genome editing...amino acids in size. To give a visual analogy: if Cas9 or Cas12a is a soccer ball, TnpB is a baseball —...genome editing, and two of our top-performing constructs are now available through Addgene (pKb-TnpB1 ...ancestors of Cas12 proteins. Functionally, while Cas9 contains two nuclease domains (HNH and RuvC-like...with a single RuvC-like domain. In simple terms, Cas9 cuts DNA using two "scissors" (one for each strand...

...Applications (CHyMErA) Knockout Libraries express hybrid Cas9-Cas12a gRNAs under a single U6 promoter. ...Figure 1:Schematic diagram of different FingR constructs and experimental results. Image from Bensussen...Bensussen et al., 2020. The FingR constructs target the synaptic scaffolding proteins PSD95 and gephyrin...excitatory and inhibitory synapses, respectively. Once expressed in the neuron, the FingRs bind to the endogenous...other regions of the cell. By combining vectors expressing different fluorescent proteins targeting inhibitory.... The team engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other cellular...proliferation in acute myeloid leukemia and saw that expression of H2B-FT consistently predicted the cell cycle...

...co-injection of the targeting vector and a Cas9 expression plasmid will insert the desired genetic tag...are then mixed and transfected along with a Cas9 expression vector. They can also be introduced directly...directly into a Drosophila S2 cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging...directed repair after a CRISPR/Cas9 induced break. Learn more about these constructs and the cell lines they’...this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a...accepts the sgRNA target sequence for U6 promoter expression and the second site accepts the homology arm ... template for inducible alteration of protein expression levels. CRISPR tagging systems offer users an...

... at Addgene  Choosing an expression system Choosing an appropriate expression vector is the first step...change gloves between constructs if you are transforming more than one construct at a time. For detailed...cause artifacts from over-expression and it is likely that such a construct will be silenced in subsequent...for visual selection of transformants based on expression of RFP in the seed (Emami, Yee and Dinneny, 2013...begins. If your plants begin to flower before your constructs are ready for transformation, you can cut off...lots of seeds. I typically use 8-10 plants per construct I transform. More seeds mean more chances to recover...generation. You also have no control over where your construct will be inserted. If your insertion occurs in ...

... need to construct plasmid vectors or synthesize/purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed...genes more effectively than Cas9 complexed with sgRNA. Direct comparison of Cas9 RNPs prepared with an in...Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease,...target site. With the widespread adoption of CRISPR/Cas9 technology and rapid improvements in the field, ...however, suffer from many pitfalls: they require construction of plasmid vectors and/or rely on cumbersome...set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope tagging in mammalian...genes at a time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging...

...light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription...proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing ...Sativa AcrIIA4 anti-CRISPR protein, co-expressed with Cas9 or dCas9 Repression of gene editing and transcription...heterodimerizing domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure...modular nature of Cas9, Nihongaki et al. showed that it was possible to swap out the Cas9 domains in their... is light-dependent (Yu et al., 2020). To express the N-Cas9, Haifeng Ye’s lab used the bacterial phytochrome...

...CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a ...different promoters driving expression of Cas9 were used in this study.  The zCas9i cloning kit is a plant ...this method, the major capsid protein VP1 is co-expressed with a fusion protein containing VP2C and a protein... of the population.  Because indel generation by Cas9 can occur at a steady rate, scientists found a way...a compact Cas9 ortholog from Staphylococcus lugdunensis that recognizes a NNGG PAM and has comparable ...with 6-12 guides per gene where gRNA expression is induced by anhydrous tetracycline. Three new anti-CRISPRs...anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein that is smaller and easier to deliver...

...cloning and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 ...Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 under the control...find more lentiviral expression vectors (empty backbones) here. Cloning and expressing new shRNA sequences...lentiviral backbone from the Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette. It can be used to...Lentiviral Expression System Kit (Kit #1000000060) The MuLE (Multiple Lentiviral Expression) system facilitates...Cre-dependent gene expression, gene knockout, and selection marker removal. CRISPR technology pdCas9-humanized ...inducible gene overexpression, knockdown (shRNA, miR-30-shRNA), mutation/editing (CRISPR-Cas9) or deletion...

...death of transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of ...research and its therapeutic potential. Note: constructs discussed are based on Adenovirus type 5. Three...capacity is limited to 8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune response... capacity (up to 36 kb!), long-term transgene expression and negligible toxicity. However, their production... cells can be broken down into 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM ...popular method for creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector ...generate the final recombinant adenoviral plasmid construct, please see Addgene’s Adenoviral Guide webpage...

...cells express iCre with a mitochondrial anchor and a TEV cleavage site. Recipient cells express DsRed ...Affordable, low-tech CRISPR/Cas9 experiments By Ashley Waldron CRISPR/Cas9 has revolutionized biotechnology...chromoprotein and gRNA plasmids. Nuclease-dead Cas9 (dCas9) protein is supplied separately. Results can ...cell-surface proteomes (Wu et al., 2025). The team expressed extracellularly-anchored horseradish peroxidase...nuclease, and excises the DsRed-STOP to trigger expression of EGFP. Figure 2: Schematic of the MitoTRACER...Confocal image of CA2 neurons in a mouse brain slice expressing PinkyCaMP; scale bar 50 µm. Reproduced from Fink...and hindbrain regions of live zebrafish larvae expressing FRCaMPi or SomaFRCaMPi in neurons; scale bars...

...pluripotent stem cell (iPS) technology and CRISPR-Cas9 constructs from Addgene to gene-edit mutations into XP...looking at a bunch of different strategies with CRISPR-Cas9. There are so many different variants and strategies...ATP1A3. The gene encodes a sodium-potassium pump expressed specifically in neurons of the brain. McSweeney... of those mutations, they now plan to use CRISPR-Cas9 technology to create cell lines and/or mouse models...ve proposed to use plasmids with modified nickase Cas9 protein in order to target the specific gene region...

...the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9 protein.... 47327 pET-28b-Cas9-His 6xHis (C terminal); NLS (C terminal) 62933 pET-Cas9-NLS-6xHis 6xHis... Find Plasmids for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression ...mice pre-CRISPR was very arduous. First, you’d construct a plasmid with your desired changes, long homology.... recommend ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components (...increase T7 promoter activity. To avoid re-cutting by Cas9 after editing, make sure that the repair template... terminal); NLS (C terminal) 62934 pET-NLS-Cas9-6xHis 6xHis (C terminal); NLS (N terminal) ...

...proteins, including Cas9 and dCas9-transcription factors, for which low expression levels are optimal....these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive... by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic...engineer new pathways with sophisticated gene expression controls. Most cell types do not respond to low-intensity... common E. coli histidine kinase. This hybrid construct senses light, and an obligate chromophore allows...only about 10-fold, precluding the study of gene expression at very high or very low levels. Schmidl et al... to better fine-tune the level of output gene expression based on the input light intensity. In addition...