...the CRISPR-Cas9 system. Mehmet Fatih Bolukbasi from Scot Wolfe's lab described a chimeric Cas9 that is improving...CRISPR-Cas9 field – Jennifer Doudna, Feng Zhang, and Keith Joung. All Addgene depositors! Addgene was mentioned...versions of each, and Tim Lu even compared his "memory" gene codes to a Turing machine (also discussed in the...harnessed. Sharing CRISPR, synbio, & more via Addgene Addgene's addition to the poster session was a first...attendant called them, "long, skinny things") and generally exuding a very-tired-but-very-excited attitude...happens): Science is hard. Chris Voigt, another Addgene depositor, discussed some of the many challenges...That so many scientists are tackling this type of genetic engineering is a testament to how amazing the downstream...

...zebrafish gene of interest? Search Addgene's collection for plasmids that contain a zebrafish gene or sequence...Lab CRISPR/Cas9-based conditional mutagenesis in zebrafish - Wenbiao Chen Lab A CRISPR/Cas9 Vector System... zinc finger nucleases, TALENS, and CRISPR/Cas9-based gene editing make the zebrafish a stalwart research...zebrafish-specific gene expression database that provides information about when genes are active in developing...developing embryos. zfRegeneration - A database for gene expression profiling during regeneration. Seurat - R ...-type specific conditional gene inactivation - Jeffrey Essner Lab GeneWeld Vectors for Targeted Integration...modified CRISPR-Cas9 system - Shuo Lin Lab Re-purposed Ac/Ds transgenic system enabling CRISPR/dCas9-interference...

... your gene of interest. For ideal use, this gRNA should be cloned into a vector containing Cas9 and the...et al. (2016).  “MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.” Nat ...harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target ...than that observed with NHEJ. PITCh: Using MMEJ for gene knock-in Building on the lab’s previous work, Sakuma...plasmic is cotransfected with a plasmid carrying Cas9, the PITCh-gRNA, and the locus-specifc gRNA. The...Contransfect the PITCh vector with the vector carrying Cas9 and the PITCh- and locus-specific gRNAs Step 4: ... is if the GFP-Puro will alter expression of the gene it follows. For increased versatility, it would ...

...-invasive systemic gene delivery in non-human primate (NHP) brains.  Systemic gene delivery capsids previously...Monomeric StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific ...peptide” that facilitates endosomal escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing...lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein (RNP) was limited (likely due ...multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization...Localization Signals, and GFP. B) Workflow to assess Cas9-PAGE editing with viral sgRNA targeting mCherry reporter...Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain....

... quantify Cas9 cleavage, thus exhibiting its versatile utility in quantifying multiple gene editing activities...reporter for rapid quantification of base-editing and Cas9 cleavage activity Article contributed by Angela ...lentiviral vector system that monitors both APOBEC- and Cas9-mediated Editing (ACE) in real-time. The reporter...Wanisch  Listen to the optogenetics podcast segment LOVTRAP is an optogenetic system that was recently...Harris lab showed that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more ... the University of North Carolina (plasmids at Addgene). It consists of two domains, LOV2 and Zdk1, where...function is more specific and much faster than any genetic or pharmacological manipulations on cells, and ...

...Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 46759 pT7-gRNA ...none, need Cas9 plasmid Chen and Wente 42250 DR274 T7 BsaI In vitro transcription none, need Cas9 plasmid...at a much lower efficiency than NHEJ. ID Plasmid Gene/Insert Vector Type Promoter PI Publication Empty...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...out our Suggest a Plasmid form or e-mail help@addgene.org to help us improve this resource!...

... In Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM = NGG) 49014 pRPR1_gRNA...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Base Edit Catalytically dead dCas9 fused ...Description Gene/Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or ... plasmid to target the dCas9-activator to your specific locus. ID Plasmid Gene/Insert Promoter Selectable... plasmid to target the dCas9-repressor to your specific locus. ID Plasmid Gene/Insert Promoter Selectable...

...explains how gene drives work and what work he's planning on doing with CRISPR-based gene drives focusing...member of the Addgene Scientific Advisory Board with a particular interest in generating Cas9 based tools...Additional Resources on the Addgene Blog Learn More About Alex's Work with Cas9 Activators Michael Koeris...developing new CRISPR tools, and the potential uses of gene drives. Listen here! Podcast breakdown (Time : ...- 31:25 : Nature's defenses against gene drives. 31:26 - 33:08 : Plans for future work at the Wyss Institute... gene drives...became a member of the Addgene advisory board where he will help shape Addgene's future. Listen to this...

...listed below. When targeting other gene loci, you will prepare the gene-specific CRISPR and donor vectors...pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh - expresses Cas9, PITCh-gRNA, & FBL-specific...Doyon lab, eSpCas9(1.1)_No_FLAG_AAVS1_T2 (Addgene #79888) , which expresses an untagged Cas9 AND a gRNA...Drosophila cells that uses PCR to generate both an expression cassette for the Cas9-programming sgRNA and HR donors...multiple genes. Plasmids can be found associated with the following article: Paix, et al. Genetics 2014 Allen...CRISPR plasmids for endogenous tagging of your gene of interest. CRISPR...repair template design. How to use CRISPR to tag your gene of interest Mendenhall and Myers Tagging System ...

...multiple terminators can serve to interrupt genes (or gene clusters) in metabolic engineering. So far,...consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing...pTargetF with the ccdB gene containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB...to successfully delete genes of interest in BL21(DE3) using the newly generated pEcCas/pEcgRNA system....used MUCICAT to optimize the gene dosage of glucose dehydrogenase and generated a strain that could achieve...J, Yang S (2015) Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl Environ...-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible sgRNA sequence targeting the pMB1...

...CRISPR/Cas9 methodology with T cell receptor engineering (Ref 5)  as described by Blue Flame Addgene depositor...using randomly integrating viral vectors, CRISPR/Cas9 genome editing allows targeted engineering of the.... "Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection." Nature 543.7643 (2017...Additional Resources on the Addgene Blog Read Additional Cancer Blog Posts Addgene's Cancer Collection Pages...this multi-pronged attack. Inflammation and tumorigenesis The conference opened with a very interesting... between tumor and immune cells in real time. Addgene depositor Professor Max Krummel from UCSF showed...poster is pTomo-Ras which has been deposited at Addgene was used to produce the glioblastoma model for ...

...Mammalian ID Plasmid Gene/Insert Promoter PI Publication Drosophila ID Plasmid Gene/Insert Promoter PI ...catalytically inactive Cas9 (dCas9) is fused to FokI nuclease. When FokI dimerizes, it generates a double-strand...PI Publication Yeast ID Plasmid Gene/Insert Promoter PI Publication Do you have suggestions for other plasmids... CRISPR dCas9-FokI CRISPR: dCas9-FokI...with dCas9 fused to FokI for creating double-strand breaks (DSBs). CRISPR...Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting RNA Editing Other...Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors gRNAs...

...CRISPR/Cas9 genetic circuits References: Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits...binding proteins with Cas9 guided by different promoters. The challenge in building genetic circuits is getting... to turn on genes at different times. It requires more complex synthetic networks. Addgene: What hurdles... that stop it from being used. One thing is that Cas9 is toxic. When cells carry CRISPR circuitry, it ...on an engineering project including hundreds of genes and regulation and resource control - everything...gives us enough hardware to control hundreds of genes. We’ve created enough regulatory elements, CRISPRs...the scale of genetic engineering, with the ultimate objective of genome design.” It’s genetic engineering...

...Resources on the Addgene Blog: Learn about CRISPR RNA Editing with REPAIR Get tips for using Cas9 Transcriptional...than other Cas13s and a whopping 33% smaller than Cas9! Their small size makes then easy to package in ...significant off-targets, CasRx displayed zero. Unlike Cas9, for which efficiency varies widely across guide...In this case, Konermann et al. targeted MAPT, the gene encoding dementia-associated tau, delivering dCasRx...possibilities. To identify Cas13d, both groups used very general bioinformatic screens that looked for a CRISPR ...regulating methods: small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and...scientific sharing will continue to grow, and we at Addgene appreciate our depositors for making their tools...

...the Addgene blog CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The ...long ago, spCas9 was the only Cas enzyme widely available and applied by researchers for gene targeting...they're available through Addgene. So whether you're looking for the exact Cas9 plasmid used by Jinek et...Resources on Addgene.org CRISPR software and resources Addgene CRISPR Guide Addgene’s CRISPR 101 eBook...and trans activity of each entry. For example, SpyCas9a (one of the most frequently used Cas enzymes) ... trans activity. AsCas12a, on the other hand, generates staggered DNA nicks at its target in cis and cleaves...end goal. CasPEDIA also features useful links to Addgene resources throughout the site. In the Experimental...

...Energy’s Joint Genome Institute for uncharacterized genes that were proximal to a CRISPR array and the universal...CRISPR-Cas systems containing cas1, cas2, cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40...-Cas systems. There are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All these...primitive version of the larger and more complex Cas9 and Cas12 proteins. Cas14 cleaves ssDNA But what...potential of Cas14, the Doudna lab cloned the cas14 gene together with the adjacent CRISPR array and intergenic... and cleave a targeted sequence of ssDNA. Unlike Cas9, Cas14 does not require a PAM sequence. In addition...presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific of a viral...

...Plasmid Gene/Insert Selectable Marker PI Publication Purify A catalytically inactive Cas9 (dCas9) can be...(HDR). ID Plasmid Gene/Insert Promoter Selectable Marker Publication Prime Edit Cas9 H840A nickase fused... to immunoprecipitate FLAG-tagged Cas9. In the CAPTURE system, Cas9 is tagged with a biotin acceptor site...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Visualize A catalytically inactive Cas9 (dCas9...dead dCas9, or dCas9 fused to a transcriptional repressor peptide like KRAB, can knock down gene expression...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or ...

...among other things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic...Cre-dependent gene expression, gene knockout, and selection marker removal. CRISPR technology pdCas9-humanized...and lentiviral vectors that are used for gene regulation, gene of interest expression, knockout, and screening...codon-optimized Cas9 under the control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown....Expressing your gene of interest pLenti-puro (Plasmid #39481) You can clone your gene of interest into...inducible gene overexpression, knockdown (shRNA, miR-30-shRNA), mutation/editing (CRISPR-Cas9) or deletion...This is a 3rd generation lentiviral backbone from the Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette...

...lab utilizes a CRISPR/Cas9 based approach to knockout, repress, and activate gene targets. To accomplish...fusion for gene activation CRISPR/Cas9 Plasmid Variants Figure 1: Schematic of the CRISPR Collection. Plasmids... plasmids dCas9-KRAB-MeCP2 fusion for gene and non-coding enhancer sequence repression dCas9-VPR fusion...CRISPR Collection The Brzezinski lab investigates gene regulation in the context of the developing mouse...taken advantage of the Zhang lab all-in-one (guide/Cas9/reporter) pX458 plasmid family. They have made several...pX458 plasmid to expand its usefulness for the lab's gene regulation and developmental studies. Key plasmid...fate specification, yet differs from deleting both genes . Development. doi: 10.1242/dev.190272. PMID: 32541005...

...technology of the year in our collection. CRISPR/Cas9 - At Addgene, 2013 will be known as the "Year of the CRISPR...frequently requested. Ubiquitin - One particular gene we saw increased interest in from 2008 to 2009 was...expression and pLVTHM, a lentiviral plasmid for mammalian gene silencing. TALENs - Genome engineering methods improved.... So far, we have distributed over 10,000 CRISPR/Cas9 plasmids to more than 3,000 labs around the world...Addgene was founded 10 years ago today. In that time, Addgene has shipped over 350,000 individual plasmids...biosensors, optogenetics, and other imaging tools. The GCaMP5 and GCaMP6 series of genetically encoded calcium...Top 10 plasmid technologies distributed through Addgene over our first 10 years. 10 most distributed plasmid...