...sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting ...genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose. Epigenetics began...many TALEN-based technologies, the advent of CRISPR has made targeting much easier! With the help of guide...modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including...BioRender.com. Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation...Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited pdCas9-DNMT3A-EGFP and pdCas9-DNMT3A-PuroR...DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion...

..., and Tol2), each of which has several different applications (mutagenesis screens, transgenic animals...excision site after cleavage by the transposase. It has a cargo capacity >100 kB, although integration efficiency...efficiency decreases with cargo size. Sleeping Beauty has a close-to-random integration profile in mammalian...hypersensitivity sites. Like Sleeping Beauty, piggyBac has an integration efficiency in human cells similar ...1996). Unlike Sleeping Beauty and piggyBac, Tol2 has a weak consensus sequence for its preferred target...hypersensitivity sites. Tol2 is only active in vertebrates and has lower efficiency of integration in human cells than... is a truncated version of the original Tol2 and has ~3-fold increase in transposition activity  Tol2...

...enzymes on the block? The Cas family of proteins has many useful genome engineering enzymes, each with...to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering purposes far ...far more than any other Cas family protein and has thus benefitted from countless rounds of optimization...effective at wiping out large regions of DNA and has anti-viral application potential as a result. Fast... Cas enzymes outlined below as well. Cas12a also has significant therapeutic promise due to the fact that...to package for therapeutic delivery by AAV, which has strict capacity limitations. New efforts have recently...for efficient, multiplexable RNA knockdown. Cas13 has also stirred up interest for its potential as a viral...

...Addgenies has helped me get a much better idea of what lies ahead. Thus the “Meet an Addgenie” Instagram interview...Director of Product Management Eric Perkins, who has been in many roles in Addgene since he started, said... set up for failure. Listening to Kenkel’s story has helped me realize that is not true at all. Racheal...medical school was devastating. At the same time, she has an entirely new path to discover and gives her time...finance, and while I've done that, my big takeaway has been the impact I can have on an organization beyond...was one of the biggest takeaways from my co-op. It has been so insightful to learn about different career... a few years ago at a CRISPR meeting.Read his Instagram feature here. You also don’t have to go back...

... expressing IgM. IgM has a pentamer structure, which means each IgM molecule has a binding capacity or...function is unknown. It has a monomer structure with a valency of two and has no subclasses. IgG IgG is... appropriate isotype control. An isotype control has the same constant heavy chain domain as your primary...replaces the hinge region in IgM antibodies. IgM has no subclasses and is found mostly in blood. IgD While...on the Addgene blog Learn about the advantages and disadvantages of using monoclonal antibodies Read ...pathogen. Different isotypes also appear at different stages of an immune response. There are three main pathogen...

...THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED ...Natronobacterium gregoryi, or NgAgo for short. Addgene has already distributed this plasmid all over the world...Publication at Addgene Cas9 and NgAgo head to head NgAgo has a number of unique features compared to Cas9. These...compare CRISPR and NgAgo, but it seems that NgAgo has lower potential for off-target effects. In Gao et...characterized Argonaute from Thermus thermophilus (TtAgo), showing that this protein could cleave DNA in ...Unfortunately, Swarts et al. were unable to show TtAgo-mediated cleavage in mammalian cells, likely due...al. searched for Argonaute orthologs similar to TtAgo and PfAgo, another thermophilic Argonaute. They ...

...Resource Center has various plasmids for overexpression and knockdown. Addgene also has a number of insect... transgene ends up (1): Random insertion has the advantage that you can get varying expression levels... 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...derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature. 472, 486–90 (2011). ...

...that for the flow plots discussed in this post, voltage has been adjusted appropriately and compensation ...interpretation. Priyamvada Prathima has a research background in protein mutagenesis and cancer immunotherapies...baseline expression of a protein while the green (top) has an upregulated expression (shift to the right). The...overlaps with the baseline suggesting this sample has a comparable expression to the baseline/negative ...adjusted voltage and compensated your colors. Here are some good practice tips on adjusting voltage: During...changing the voltage for the particular fluorophore prior to recording data. Increasing the voltage will shift...decreasing the voltage will shift it to the left. If your signal is too high, lower the voltage to have your...

...approaches, has its own unique set of obstacles. Thus far, several targeted therapies have taken advantage of ...There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field ...CRISPR Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer treatment...CRISPR Therapeutics. Data shared from these trials has been outstanding, with almost all patients experiencing...desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One treatment...delivery tends to be trafficked to the liver. Thus, it has been used for hATTR therapy, since TTR is primarily...targeting the diseased allele specifically. Early-stage trials showed a high success rate in clearing misfolded...

...diverse bacteria from various environments. It has the advantage of stable genomic integration and is easily...Flp recombinases used to excise markers. It has the advantage that CRISPR efficiency can be controlled by...Cas9-adenosine deaminase fusion Caixia Gao’s lab has developed a new plant base editor from an evolved...String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR), developed by Stefan Stricker...high numbers of gRNAs in one overnight reaction. STAgR is highly customizable as vector backbones, and ...chosen. Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows CRISPRi...

...to define how you will know that the relationship has been successful – what are the desired outcomes? ... Dora Farkas, founder of Finish Your Thesis (who has also blogged for us) offers a free report on her ...called “Secrets to Success in Graduate School”. She has also launched an online career coaching program that...in Madison, Wisconsin she also works remotely and has experience as a scientific recruiter. Chris Humphrey...of From PhD to Life. She runs great tweet chats, has an outstanding blog and, while her PhD is in History...remotely, will travel for speaking engagements and has many areas of specialty from leadership development... consultation meeting or call for free.  Take advantage of this to get the information you need to choose...

... The key advantage of using RNA to reprogram cells is that it’s simple and efficient. It has a high efficiency... field of induced pluripotent stem cells (iPSCs) has been around for 10 years. In that time, scientists... of Myc or other oncogenes after differentiation has been linked to tumor formation in iPSC-derived and...iPSCs compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic...factors driven by one promoter. While lentivirus has similar rates of reprogramming when compared to MMLV-derived...mechanism between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected...cell division. A drawback to this approach is it has lower levels of efficiency at generating iPSCs, usually...

...methods. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful...of potential clones. Golden Gate assembly has a few advantages over other cloning methods. Exonuclease-...just a few steps. More recently, CRISPR technology has adapted Golden Gate cloning for inserting the appropriate...also be adapted to express multiple gRNAs. Addgene has two Golden Gate-based gRNA assembly methods available...added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning Golden Gate cloning is...destination vector, making multiplexing easy.  Disadvantages of Golden Gate cloning Golden Gate cloning is...

...agent than BME and has a less… distinctive smell. BME smells like rotting eggs but has a longer shelf life... It doesn’t degrade during long-term storage and has both high binding capacity and high protein retention... specialty low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes... other hand, doesn’t require any pre-wetting and has lower background than PVDF. It is a little quicker...quicker to use, with high binding capacity, though it has lower retention after binding and washes. It’s less...and need to be read fairly soon after the reaction has started. Types of chemiluminescence enzymes The two...darkroom and a developer. Film is highly sensitive but has a limited dynamic range, which means it may take ...

... an essential aspect of biological research that has defined our understanding of biological processes...model organisms is Escherichia coli. The bacterium has been used as a model organism for over 60 years with...cloning and metabolic engineering.  The E. coli genome has an extensive research history and characterization...studies. For example the E. coli biotin ligase, BirA, has been used to identify and study protein-protein interactions...APEX (Rhee et al., 2013). Research using E. coli has the potential to address many areas of microbial ...research with an archive of information and data that has yet to be produced with mice (Iannaccone et al., ..., 2009). While the rat as a model organism has its perks, it is far from perfect. One of the most limiting...

...technology has been studied and used in assays for over half a century; the Addgene blog has an excellent...new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium...initially still unstable in eukaryotes, the Jaffrey lab has since developed new RNA-based scaffolds to contain... As biosensors, aptamers hold a few important advantages over their amino acid-based counterparts, antibodies...acceptor FRET know-how with aptamers, aptabeacons (or aptatags) were born. Aptabeacons can be created in many...quenchers are still relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity... Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the nature of GFP fluorescence...

...Repair has been viewed 219,289 times, while Validating Your Genome Edit has 76,011 views.  Has all of ...libraries Addgene has distributed (1C), from 2012 to 2023. Addgene’s collection of CRISPR tools has grown steadily...2023, our CRISPR guide has been viewed 1,904,462 times, and the CRISPR 101 eBook has been downloaded over...globe. Our first story focuses on a technology that has changed virtually all of biology…CRISPR.  Since ...own to share. Over the years, Addgene’s repository has grown to include many CRISPR tools.  Distributing... shipped out each year.     Figure 3: Percentage of requests per year for CRISPR plasmids categorized...

...graduate program at the University of Manitoba. Our lab has been utilizing chemogenetics (Designer Receptors ...Although AAV2 can transduce many tissue types, it has low expression in several brain regions where other...serotypes to infect different cell types. In the CNS, it has been shown that there is variable transduction of...from the Original Location - Axonal Transport It has been demonstrated that certain serotypes can undergo... of movement, distance traveled, etc.) (4). This has also been identified with AAV2 (5), AAV6 (6) and ...required for the onset of gene expression. However, it has yet to be determined what gene expression looks like...problem in many experiments, a group of researchers has identified this feature of AAVs as a new tool for...

...studied but it is only the tip of the iceberg as it has been predicted that many others are still unknown...enzymes. In contrast to other Cas enzymes, Cas14 has not been found in bacterial genomes but only in the...add-on to a diagnostic toolkit, called DETECTR, which has been created by the authors. This kit uses Cas12 ... Cas systems, Jennifer Doudna’s lab analyzed metagenomic datasets to try to determine whether simpler ...they mined a database of microbial genome and metagenome from the Department of Energy’s Joint Genome ...

...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...The risk of generating RCL is low but extra care has to be taken especially if you are working with proto-oncogenes...previously used to drive expression from the LTRs) has been removed, Rev (which facilitates nuclear export...expression. In contrast, in the Tet-on system, tTA has been modified (and renamed rtTA) to bind TRE only...viral binding. This method, called spinoculation, has been found very effective for T cells transduction...to infect your cells of interest.   Advantages Disadvantages Can carry large transgenes (>8 kb) Potential... cells, it was obvious that its safety was a disadvantage particularly for clinical development. In addition...