...isoform-specific gene expression. Gene isoforms refer to mRNAs generated from the same gene but with different...variant of Cas9 (dCas9) and engineered combinations of transcriptional activators to induce gene transcription...isoform-specific gene regulation — at least, more straightforward than generating cDNA for each gene isoform individually...first ask yourself whether gene overexpression is necessary (instead of gene knockout or knockdown, for...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church... neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation." eNeuro 6.1 ...transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice." Nature...

... - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated...the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion Caixia Gao’s lab has ...tRNA adenosine deaminase that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits... a few of the new CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR... in Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA ...is a single step gRNA multiplexing system that generates vectors containing high numbers of gRNAs in one...a new CRISPR tool you’ve recently deposited to Addgene and you’d like it to be included in the next What...

...domain due to a gene that resembles cas10. All type IV subtypes are missing adaptation genes, Cas1 and Cas2...–C, with the beloved SpCas9 (from Streptococcus pyogenes) belonging in II-A. Cas9 was the first to be ...sequence similarity, phylogenetic analysis, neighborhood analysis of surrounding genes, and experimental ...first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied interest in...straightforward as it seems. There is not a single gene that is shared across all CRISPR types; the fast...while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out the...together because they contain a distinct Cas7-type gene. The exact mechanism of type IV systems is not known...

...hammer: Gene knockout by NHEJ Gene knockout with CRISPR technology is usually accomplished by Cas9-mediated...technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter of a target gene. Here, the target window...you are trying to do: gene knockout, a specific base edit, or modulation of gene expression. Location ...multiple sequences targeting a gene to score in order to call that gene a hit.  This is an exciting time...Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets. Nat Commun 10: . https:/...protein-coding capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG...This means that, for gene editing, there are usually few potential gRNAs. While SpCas9, with a PAM preference...

...editing technique that relies on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing...., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure...pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created ...along with your intended edit. This prevents the Cas9 nickase from re-binding and nicking the newly synthesized...Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, ...and goal of the experiment. Now, onto the tips! General principles pegRNA design If you’re just starting...programs to help you design pegRNAs. Here on the Addgene blog, we featured a guest blog by the developer...

...the dramatic spread of the Cas9 technology: "The rapid adoption of the Cas9 technology was also greatly...program are here. This was a great opportunity for Addgene to present our own data on plasmid deposits and...and distirbution for these fast moving fields.  Addgene is a global nonprofit plasmid repository. Over ...Over 2,000 labs have deposited plasmids to Addgene and we distribute over 130,000 plasmids in 2014. Thus,...combination of open-source distributors such as Addgene, as well as a number of online user forums such...Meeting Eric presented a poster with data comparing Addgene global deposit and distribution trends for Zinc...

... on therapeutic strategies that use gene replacement therapy, gene editing, MECP2 reactivation, RNA editing...Novartis and rebranded to Novartis Gene Therapies, and Taysha Gene Therapies, founded on Gray’s work, ...corrected by CRISPR/Cas9 in wild-type human iPSCs. Image modified from Le et al., 2019. Gene editing via ...the CRISPR/Cas9 system to both precisely insert the MECP2-R270X mutation into the MECP2 gene in wild-type...protein 2 (MECP2) gene, Rett Syndrome is a rare, progressive, neurodevelopmental genetic disorder that affects... CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells. Front Genet 10: . https...of Rett Syndrome research, the role of the MECP2 gene, and highlights some of the latest breakthrough ...

...critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage... introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-...resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used ...footnote; a similar approach can be used to identify Cas9/CRISPR-induced genome modifications. Occasionally... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate...modifications.   Site-directed mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers...

...using bacterial phage vectors with CRISPR/cas9 sensitizing gene cassettes. Thse vectors are engineered to... tetracycline cas9 inducible knock-out system. His lab is also exploring the use of cas9 proteins from...were identified for 61 such diseases. Now single gene alleles have been identified for 4,163, over 50%...Lowe talked about his work on the 500+ human tRNA genes and, until now, we've overlooked regulatory fragments...may be involved in preventing silencing of self-genes. Dr. Mello presented a hypothesis that these pathways...involved in RNA based surveillance of repetitive gene families and provide a way to guide inheritance ... to replace the drug resistantance genes in the bacteria thus making the bacteria sensitive to known antibotics...

...engineered Cas9 variant might work for your experiment. PITChing MMEJ as an Alternative Route for Gene Editing...Corn’s lab describe how a better understanding of Cas9 biophysics can help enhance HDR. CRISPR 101: Ribonucleoprotein...tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports over 250 genomes, and...template to increase efficiency of insertion and gene replacement. Care to share your favorite CRISPR ...Blugene and I represented Addgene at the recent Keystone meeting on Precision Genome Editing with Programmable...hopes to evaluate genome editing pipelines and generate a common lexicon for genome editing studies. These...desired edit, and I want to make sure that I (and Addgene) don’t forget this in the future. Here’s a list...

...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...

... 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 ...

...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...

...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 ...

...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...