...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...

...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...

... 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 ...

...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...

... 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...

...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...

...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...

...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...

...resources include plasmids expressing Cas9, empty gRNA backbones, and constructs for endogenous tagging. ... genes by feeding worms bacteria expressing dsRNA or use CRISPR/Cas9 gene editing to easily engineer specific...characterized which allow researchers to express desired constructs in precise cell types and/or developmental... via a CRISPR-Cas9 system. Cloning-free CRISPR for C. elegans , which uses purified Cas9, such as that...that expressed from pHO4d-Cas9 from Michael Nonet. Protocol can be found at the (Link opens in a new window...and spatial control of gene expression in C. elegans by combining expression of the FLP recombinase from...Worm Plasmids and Resources Plasmid Tools Worm Constructs Kits and Collections Other Resources The nematode...

...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...

...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...display increased neuronal expression in the brain while having decreased expression in the liver after systemic...only AAV.CAP-B10 causes brain wide expression, with no liver expression, after retro-orbital sinus vein ...Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced...10.1038/s41593-021-00969-4.   Increased neuronal expression using Cre-mediated AAV Targeted Evolution method...vein injection of a CAG-driven mNeonGreen construct in mouse. Image from Goertsen D, et al., 2022.  Find...of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large inserts. However, this system ...

...full selection of plasmids expressing different functional variants of Cas9 and empty gRNA backbones. ... assembly of one or more gRNA expression cassettes - Qi-Jun Chen. Cas9 and gRNA backbone for use in rice...DNA modules for assembly of CRISPR/Cas9 constructs - Sylvestre Marillonnet and Johannes Stuttmann. Combined...CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner. CRISPR-Cas9 vector...creating plant expression vectors: A modular plasmid assembly kit for multigene expression, gene silencing...create plant expression vectors containing several cassettes and generate multi-construct transgenic plants... - Tools for gene and protein expression and mapping of gene expression in plants. Figshare - Plant Illustrations...

...lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself...Addgene makes it so much easier to share your constructs from lab to lab. Promega supports the Addgene...endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc is very inviting for endogenous...only 19 kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined...fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged into a retrovirus...reporter was used to sort successfully infected/expressing cells by flow cytometry. The isolated cells were...reporter can improve deep tissue imaging. The constructs for GpNLuc and OgNLuc are available through Addgene...