...wildtype Cas9 with optimized chimeric gRNA has high efficiency but has been shown to have off-target effects... the Genome Engineering Using CRISPR/Cas Systems has expanded out of control. To further consolidate these... a new system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design...recombination? A3: The most we've tried to insert so far has been 1kb. We used homology arms that were 800bp long...for positive ones.  -- OR -- If your HR template has a selection marker such as puromycin, you can (also...

... requested ubiquitin plasmids were pRK5-HA-Ubiquitin-WT and HA-Ubiquitin, and we now have 19 different...founded 10 years ago today. In that time, Addgene has shipped over 350,000 individual plasmids to 5,000...5,000 different research institutions. This has given us a unique window into technology trends in the life...the CRISPR." The newest genome editing technology has been used and modified by so many labs and for so...

...and divide along with the endogenous DNA. AAV also has low immunogenicity, meaning it does not induce the...conducive to targeting all cell types. Altogether, AAV has many excellent features for human gene therapy and...administration, such as in mouse experiments. AAV also has multiple serotypes (12 naturally occurring and more...constraints are an issue. Adenovirus Adenovirus has a linear, double stranded DNA genome which is non-enveloped...cancer in hamsters, although no cancer-connection has been identified in humans to date. Over fifty serotypes...have been identified so far! Adenovirus generally has high immunogenicity, but some strains have been engineered...other viruses discussed in this article, adenovirus has the highest maximum titer, a plus for production ...

...Resource Center has various plasmids for overexpression and knockdown. Addgene also has a number of insect...locus your transgene ends up (1): Random insertion has the advantage that you can get varying expression... to determine in which chromosome your transgene has integrated. This can be accomplished with a few matings... embryos and pick out flies where your transgene has been incorporated into the germline. Outsourcing ... have to map into which chromosome the transgene has integrated and make genetic crosses with balancer...finishing his immunology PhD at Harvard University. He has had nearly a decade of experience working with Drosophila...

...lab at the Massachusetts Institute of Technology has been “working on new experimental and theoretical...and medicine. As they’ve gone along, Voigt’s group has also been assembling the toolbox needed for anyone...CRISPR circuitry, it can slow their growth. Something has to be done about that.  The second problem is fundamental...this now, we have ideas, but so far nothing that has worked. I don’t have the solution. Our paper shows...transcriptional and translational activity a cell has and control how much it taxes the resources of a ...antibodies. But we are really just using what biology has given us. Wood is a complicated material and we can...can access it, but we are stuck with what biology has given us. Over the next 100 years, we are really ...

...CRISPR has greatly enhanced the ability of scientists to make genomic alterations, bringing about a revolution...inserting a custom gRNA.  While this methodology has proven to be incredibly valuable to scientists, there... used to deliver a Cas9-gRNA RNP. This advantage has allowed scientists to perform in vitro and in vivo...to the use of this technique in cell culture, it has also been applied to genome editing of mouse zygotes...the early stages of development. David Liu’s Lab has demonstrated the use of cationic lipid-mediated methods...et al. 2015). A more targeted approach using RNPs has recently been described in vitro, with the hope of..., as an alternative to plasmid based techniques, has been an area of intense study. Two methods to accomplish...

...CRISPR/Cas9 has made it easier than ever to make precise, targeted genome modifications. Cas9 has been modified...which one of Cas9’s two critical enzymatic residues has been converted to an alanine (D10A or H840A), changing... for your experiment, ensure your sgRNA sequence has little homology to other regions of the genome and...looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement of NG, GAA, or GAT, making it a...SpG variant, discovered by the Kleinstiver group has a PAM of NGN (Walton et al., 2020). Further, this...reverse transcriptase, and a nCas9. While this system has been shown to be less efficient than base editing...directly fusing the cargo to dCas9.  This technique has been exploited to localize factors such as transcriptional...

...give me a brief history of optogenetics? How long has this idea been around? Boyden: Optogenetics is a ... silencers of neural activity. Since then, there has been a steady stream of papers describing new photosynthetic...against each other. Getting two-color stimulation has been a holy grail of the field. The problem is that...is fundamental understanding of the brain, which has humanistic and philosophical implications. As an ...Addgene over the years, many of them very popular. Why has it been so important to you to distribute these tools...as possible? Boyden: I think because neuroscience has so many different goals: to understand how a mouse...

... to be activated by the DREADD actuator after it has been administered. Although we found a larger proportion...DREADD to the plasma membrane. The haemagglutinin (HA) tag is a small 9 amino acid epitope of the haemagglutinin...al., 1988). Thus, we injected AAVs carrying hM4Di-HA in the brains of mice and monkeys. Surprisingly, ... a big difference. Replacing the mCherry with the HA tag resulted in abundant expression of hM4Di in plasma... where the fluorescence is not needed, the use of HA and other short epitope tags (Kimple et al., 2013...used to reveal the hM4Di coupled to mCherry (top) or HA tag (bottom). The images show examples of dendrites... to the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di...

...both a fundamental driver of these principles and has emerged as a byproduct of connectivity. Between 2012...integrated with most of the tools noted above, and has a modern API. Integrations with additional tools ..., so it’s possible to re-export a protocol which has been edited back to protocols.io. Thus, RSpace becomes...Figshare. Any ELN that facilitates reproducibility has to make it easy to export data from the ELN directly... these five general purpose repositories. RSpace has been integrated with two of the five: Figshare and...additional metadata in the repository after the deposit has been made. It’s possible to associate the depositor...the repository. The ability to deposit data that has already been structured in folders or notebooks in...

... a family affair back in 2004. As Addgene has grown, it has taken effort to continue the tradition and...goofing off, the company culture here at Addgene has remained paramount. Candidates who interview at Addgene...testament to the familial aspect of Addgene. As our size has drastically increased, it would have been a tight..., even for an event where there is food and beer. Ha! We had over 60 people join the fun - including a... or binging a new Netflix series. The thing that has made all the difference here at Addgene is the emphasis...and a cooler. Chances are one of your colleagues has a frisbee at least. Use it! 8. Organize If you’re...there is something fun happening! Addgene actually has chimes outside of our break room. A ring of the chimes...

...which a good antibody has not been developed (and just because your protein has a commercially available...localization, or protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins,...affinity tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of the ...Purification Good for antibody-based purification; has inherent enterokinase cleavage site GST Large Protein...against proteolysis, but may reduce solubililty HA YPYDVPDYA or YAYDVPDYA or YDVPDYASL  1.1 Affinity... a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass several other tag combinations...necessarily want them to stick around after that purpose has been served. At this point, proteases can be your...

...Alternatively, you can select a target sequence which has already been shown to work in CRISPR experiments....experiments. Addgene’s dedication to resource sharing has led us to develop a searchable and sortable datatable ... to the fact that every gRNA listed in the table has been used effectively in a published article. This...target sequence, and model system. Even if a gRNA has been demonstrated to work in your system, it is worth...that locus. The data in the validated gRNA table has been derived from scientists’ submissions, either...

...artifacts, it has better folding and maturation kinetics than other dark fluorescent proteins, it has good signal...Technology (KAIST) and Institute for Basic Science (IBS) has developed another useful optogenetic tool that enables...facilitate future work with this goal, the Barrick lab has deposited the pDCAF3-IS5 plasmid containing a synthetic...operon originates from Pseudomonas putida CBB5 and has been tailored for expression in E. coli. This operon... contributed by Christina Mork The Murakoshi lab has developed two new FLIM-FRET acceptors, ShadowG, a... due to its very low quantum efficiency. ShadowY has a lower quantum yield and higher absorption than ...

... world has battled the COVID-19 pandemic over the past few months, the scientific community has seen a...research into this disease. A key reason this research has been able to progress so rapidly is scientific sharing...is applicable to researchers worldwide. “COVID-19 has spread incredibly fast and without any notion of ...COVID-19 Resources Canada site. To date, the portal has recruited 3,218 Canadian volunteers, and features...

...story, to get scientists excited about what Addgene has to offer, and to help scientists find what they need...homepage. Blugene, our trusted friend and mascot, has undergone a make-over as well. While in the course...Blugene needed the same thoughtful treatment. Blugene has been with us since 2011, and while you may have seen...and sharing.  One big feature upgrade? Blugene now has a full expression. You’ll mostly see a friendly smile...an experiment doesn’t go as planned. Blugene also has full arms so that they can hold and hug items closer...

... Even though GFP has been so thoroughly characterized, it turns out this protein has a few more secrets...photoconvert GFP from green to RED! This simple technique has been shown to work in plant, Drosophila and mammalian...cases, however, a GFP-tagged cell line or organism has already been made, and it would be expensive and ...is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants used by Sattarzadeh...

... even society. One obvious way to do that is and has been to publish in journals with a high “impact factor...cited articles appearing in lower-prestige journals has risen steadily from 1995 to 2013. Considering citations...online access on scientific impact Partly, the story has to do with online accessibility. It is now about ...among the most cited. And, as scientific discourse has increasingly moved online, surely times have changed...

... how a plasmid has been used by multiple labs and see what experimental systems it has been validated ...validated in.  If a plasmid's Addgene ID # has been referenced in other publications, you'll find a link to the... and the PubMed ID # (PMID) for the article that has used this plasmid. Happy researching! If you need...

...   By Ashley Waldron Proximity-dependent labeling has become a powerful approach for proteomic mapping ...our 2019 overview). Until recently, the approach has been hampered by the need to tag each protein of ...M-CREATE) by: Brian O'Neill  The Viviana Gradinaru Lab has previously developed the multiplexed version of the...sequence knock-ins by: Lucie Wilson Le Cong’s lab has developed a method using dCas9-SSAP to knock-in long... methods for large inserts. However, this system has a much lower error rate. For a one kilobase insertion... its functions. Recently, Ravid Straussman’s lab has developed visual barcodes for clonal multiplexing...