...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...They’ve deposited plasmids for active and inactive CasX for E. coli and human expression. Read the paper...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated...Nucleic Acids Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition library...vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins of fluorescent...

... enzyme with a fully active Cas9 enzyme. PE-Cas9 comprises an active Cas9 fused to the reverse transcriptase...restoring expression in the liver.  Read the paper in Nature Biotechnology Find the PEDAR plasmids   Additional...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids Topics: CRISPR, Other CRISPR... where we highlight a few of the newest CRISPR plasmids available at Addgene. If you want to read more...will cover: Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas...drawback to the commonly used S. pyogenes CRISPR/Cas9 system is that it is too large to be effectively...CasMINI system is less than half the size of the Cas9 or Cas12a based systems, making it well suited for...

...control gene expression. Get the full scoop from Addgenie A. Max Juchheim in this post. 3. Plasmids 101: E.... the strains you can use to express your protein of interest. 4. Plasmids 101: Blue-White Screening Getting...While the vast majority of CRISPR plasmids in our repository use Cas9 to cut DNA, Addgene Science Communications...CRISPR mediated genome engineering. 10. Plasmids 101: Control Plasmids Need help thinking about the types ...Scientists eBook Download the Plasmids 101 3rd Ed. eBook Read the Most Recent Plasmids 101, CRISPR 101, and Career... two eBooks (Management for Scientists and the Plasmids 101 2nd ed, the 3rd edition has now been released...top of the many developments in science and the plasmids, technologies, and researchers behind them. As...

...preps for the delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select gRNAs...accelerate your research.   Why distribute virus? Plasmid distribution is booming, but, over the years, many...list of what’s available or suggest additional plasmids with the Viral Service Suggestion Form. Lentivirus... to knockout a gene, replace it, modulate its expression, or perform a genome-wide screen, we’ve got viruses...experiments, while rtTA can be used to activate expression of a gene under the control of the tet operator...page and includes western blots as well as other expression data. If you have any questions about the Viral...Uses and Overview AAV: A Versatile Tool for Gene Expression in Mammals Additional Resources on Addgene.org...

...combining specific sgRNAs with Cas9 or Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks... 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...) . This tracrRNA was combined with the sgRNA expression vector allowing the screen to be conducted with...

...deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding repertoire of...bound to the DNA. Cas3 or Cas9? Large genomic deletions can be achieved using Cas9, but Cas3 may be a better...previously mentioned issues with expressing all Cascade components can make Cas9 easier to work with than Cas3...its partner in crime, Cascade. Find Cascade-Cas3 plasmids in Addgene's collection! A “complex” system Cas3...challenging. It can be difficult to ensure comparable expression of all components of Cascade for maximum efficiency... Various labs have designed all-in-one Cascade plasmids — such as the Tomoji Mashimo lab and the Tilmann...efficiency of these systems, though consistent expression can still be a challenge. A multitasking Cas ...

... a GAL-4 based bipartite expression system that can regulate gene expression in C.elegans. In this system... cGAL can drive expression of your gene of interest.  SapTrap Tool Kit for CRISPR Cas9 edits At Addgene... CRISPR plasmid can be created in a single tube. You can also find additional SapTrap plasmids used for...about C. elegans and the plasmids we have at Addgene check out our worm expression resources page. This page...about SapTrap Read about CRISPR/Cas9 in C. elegans Browse our Plasmids 101 blog posts Resources on Addgene.org...CRISPR techniques and new tools for conditional expression and degradation. These workshops not only highlighted...discussed at the meeting, in addition to useful plasmids and toolkits available at Addgene.  C. elegans...

... smaller size of the plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system...have previously been stably transfected for Cas9 expression. The Yusa lab has used their library to screen...It’s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and ... thousands of targets at once. That’s what our plasmids allow.” Wang’s library is designed to target a...Lander library uses sequential infection first of Cas9, then with the sgRNAs, and can result in a higher...application offer proof-of-principle that CRISPR-Cas9 can be used to target any sequence in the genome... Genetic Screens in Human Cells Using the CRISPR/Cas9 System. Wang T, Wei JJ, Sabatini DM, Lander ES. ...

...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ...facilitates endosomal escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing of 80–90% in ...all applications. Addgene’s plasmid collection includes over 10,000 plasmids with a FLAG tag (!), but did...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... al. 2023 with permission.   Find Cas-PAGE plasmids here!   Zhang, Z., et al. (2023). Efficient engineering...

... to use variants of Cas9, like split Cas9, or a smaller Cas9 ortholog like SaCas9. You could even venture...to use a multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the other...Addgene. The large packaging capacity and stable expression of lentiviral vectors make them an attractive...attractive option. However, safety requirements and expression limitations may be too constraining for some experiments...improved safety profile, customization, and enhanced expression in many mouse tissues. Safety AAV and lentivirus...significant degree using different types of AAV. Expression Another advantage of AAV vectors is how the cargo...cargo — in this case, the CRISPR components — are expressed. Unlike lentivirus, cargo delivered by AAV does...

... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...states. dCas9 gets a colorful makeover Even without its nuclease activity, catalytically dead Cas9 (dCas9... colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While...hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make... chromatin organization. This fluorescent CRISPR/Cas9-based method is a great step forward in live cell...As this study joins the pantheon of useful CRISPR/Cas9 techniques, we at Addgene are excited to see what....1420024112. Epub 2015 Feb 23. Pubmed. Find the plasmids from this publication at Addgene. Dynamic imaging...

...Silvana Konermann, the first generation of CRISPR-Cas9 plasmids were not designed with gene activation in mind...tetraloop and stem-loop 2; 3. The MS2-P65-HSF1 plasmid which expresses the activation helper protein. SAM can ...question for her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that...describe just such a system, which they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an... three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA...system, Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use...levels of control in the system. First of all, the Cas9s can be turned on and off, to reversibly activate...

...Postdoctoral Researcher in Jacob Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts...replacement by HDR. Understanding Cas9 biophysics enhances HDR We first measured Cas9 dissociation rates and discovered...of reagents is relatively modest, requiring expressed Cas9 protein, transcribed sgRNA, and optional donor...interaction with the Cas9-DNA duplex. Image from Richardson et al., 2016.   dCas9 enables sequence replacement...normally accompanies Cas9 cutting. We still don’t know the mechanism underlying dCas9 editing, but it could... cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors...factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay...

...catalytically active Cas9, the short, CRISPRainbow gRNA will permit labeling, but a newly expressed standard length...PMC4864854. Find the plasmids from this publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA engineering... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its... All 6 gRNAs can be expressed in a single vector, pCRISPRainbow-DONOR1, with dCas9 supplied by a separate...success of Ma et al.’s approach, fluorescently labeled Cas9’s come with a number of limitations. Since color...by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence must be located...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...about a recent plasmid deposit please sign up here.   Listen this episode of Hot Plasmids! Light controls...available in a mammalian expression vector (pcDNA3) and a bacterial expression vector (pRSETB). Fabritius...APOBEC- and Cas9-mediated Editing (ACE) in real-time. The reporter constitutively expresses eGFP to indicate...Hahn's lab at the University of North Carolina (plasmids at Addgene). It consists of two domains, LOV2 ... the same number of fluorophores. You can find plasmids containing the cobalamin binding riboswitches ...

... 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active at high...used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity ...Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while GeoCas9 has maximum...bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures...discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR/Cas9 genome...discovered a Cas9 protein from the thermophilic bacteria Geobacillus stearothermophilus (GeoCas9). G. stearothermophilus...human plasma. The discovery of GeoCas9 clears the way for CRISPR/Cas9 genome editing in facultative and...

...needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read...CRISPR system. Like Cas9, Cpf1 contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease...Potential advantages of Cpf1 over Cas9 Type II CRISPR systems based on Cas9 were thought to be the simplest...than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency...rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts...display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like...

...with all the new plasmids. So far, we have distributed over 10,000 CRISPR/Cas9 plasmids to more than 3,...for shRNA expression, such as pLKO.1 puro and pLKO.1 – TRC cloning vector. Lentiviral Plasmids -  If you're...packaging and expression vectors, including pMD2.G and psPAX2. Actually, these two plasmids from Didier ...influx of plasmids for reprogramming differentiated cells into pluripotent stem cells. Plasmids from Shinya... have 19 different ubiquitin plasmids in the repository. 100,000 Plasmids Shipped! - By 2010, Addgene ...our stride. We shipped our 100,000th plasmid. A variety of plasmids continued to be popular years after...as pBABE-puro for retroviral expression and pLVTHM, a lentiviral plasmid for mammalian gene silencing....

...Yonglong Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen and colleagues...tested is expressed along with Cas9. If the gRNA effectively cuts the target sequence, the plasmid undergoes... New CRISPR plasmids available! Interested in purifying Cas9? Check out pET-28b-Cas9-His from Alex ...Cre-based Brainbow-expressing cells. Autobow plasmids are all-in-one versions which express self-excising ... host cells in order to optimize expression. The expression plasmids for a given species are based on ... Hot Plasmid list. Other popular plasmids & blog posts @Addgene: The 10 Most Distributed Plasmid Technologies...colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain...

...changes we’ve made: New CRISPR plasmid pages We’ve given our CRISPR Plasmids and Resources a fresh new design...design, sorting the plasmids we have available by category. Each plasmid page now has a helpful graphic...introduce you to the plasmid class covered on that page. To help you find popular plasmids, we’ve also added... new plasmid pages that cover some of the hottest CRISPR tools. Base Edit: Base editing plasmids allow...Antimicrobials Day 7: Multiplexing and Cas9 variants: CRISPR 101: Multiplex Expression of gRNAs and The PAM Requirement...libraries: Genome-Wide Screening Using CRISPR/Cas9 and Plasmids 101: NGS Quality Control for Pooled Libraries...revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that’s not our only job - we strive to also...

...pool of lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid libraries containing...multiplexed gRNA expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic...and ligation steps to build barcoded lentiviral plasmids containing one or more gRNAs. As each ligation...containing combinations of different gRNAs in each plasmid. Each gRNA combination can be tracked and quantitatively...activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. ...PMC4559080. 8. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166...

... 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,...on the Addgene Blog Plasmids for endogenous gene tagging in human cells Plasmids 101: Protein Tags Fluorescent...Addgene.org Browse CRISPR plasmids Check out our CRISPR guide pages Find ready-to-use Cas9 lentivirus ...target site. With the widespread adoption of CRISPR/Cas9 technology and rapid improvements in the field, ...from many pitfalls: they require construction of plasmid vectors and/or rely on cumbersome in vitro synthesis...

...recruit transcription factors to increase gene expression. CRISPRa methods vary in their transcriptional...chains, which are relatively large and are not expressed consistently throughout cells. VPR Description...Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic...J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods...Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods 13:563...transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038...Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9–based transcription factors. Nat Methods 10:973–...

...of the new plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles.  ...2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane protein (Plasmid #141386-LV [+M], negative...Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi...using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi ... over the original CROPseq. Find CROPseq-multi plasmids here Walton, R.T., Qin, Y., Blainey, P.C. (2024...Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of ...end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism for engineered...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion Caixia... paper in Science Find the anti-CRISPR expression plasmids CRISPR-based method for cell lineage tracing...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated...Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA cloning (STAgR...tRNA adenosine deaminase that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits...

...effects of gRNA and Cas9 expression, they fail to account for the effects of Cas9-induced dsDNA breaks... that either stably expressed Cas9 or that had been lentiviral infected with Cas9. Validating the Library...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved.... Unanswered questions about genome-wide CRISPR/Cas9 screen design Are non-targeting guides an appropriate...single-guide studies? Many strategies exist for reducing Cas9 off-target effects, but most studies have focused...previously identified essential genes while previous Cas9 and shRNA library screens only identified  60% of...screen. This toxicity was used a proxy for detecting Cas9 cutting. Safe targeting guides better control for...

...of the second plasmid can be done in one day in one assembly reaction. This second plasmid saves you a ...alerts when plasmids become available.  We’ve also had some requests for variations on the plasmids we’ve developed...and Myers for speaking with us! Find the Plasmids: Plasmids for protein tagging with CRISPR Additional...efficiently, they’ve recently adapted the CRISPR/Cas9 system for protein tagging. We spoke with Mendenhall... available at Addgene. For more details on the plasmids that are currently available along with detailed...several projects and this is one related to gene expression. One goal of the ENCODE project is to look at...system, what we are doing is adding in a second plasmid to target an epitope tag onto the transcription...

...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...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 ...mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP) which insert...Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced...

...off-target editing based on Cas9 off-target activity.  Find CRISPR base editing plasmids at Addgene Improving..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base...et al., 2020 SpRY Cas9, available fused to ABEmax(7.10) or to BE4max   Evolved SpCas9 with NRN or NYN PAM... 1.1%. These numbers are a vast improvement over Cas9-mediated HDR for the loci tested, where average ...using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida et al., 2016). Target-AID acts ...

...This PITCh plasmic is cotransfected with a plasmid carrying Cas9, the PITCh-gRNA, and the locus-specifc gRNA...containing Cas9 and the PITCh-gRNA. Step 3: Contransfect the PITCh vector with the vector carrying Cas9 and ... The ready-made PITCh plasmids available from Addgene are perfect for expressing GFP from a given promoter...CRISPR-Cas9 with the PITCh systems.” Nat Protoc. 11(1): 118-33. PMID: 26678082. Find the plasmids from ...Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with Us  ...Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about... knock-ins, some labs have used NHEJ for whole plasmid integration; however, since NHEJ is error-prone...