...translational termination features with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette...knockout – conversely, putting the loxP sites on either side of a gene (called “floxing”, for “flanked by loxP...experimental constraints. This has been used for a wide range of purposes, including activating an oncogene...switch recombination at individual switch regions evidenced through Cre–loxP–mediated gene targeting. Gu, ...

...rescued by zygote co-injection of Cas9 and a single-guide RNA targeting the mutant allele. When all was said...hATTR) hATTR is caused by a variety of single nucleotide polymorphisms (SNPs) in the TTR gene and is molecularly...Off-target effects are also important safety considerations for all CRISPR therapies, but especially concerning...CRISPR therapies have made significant clinical strides since we first wrote this blog post. The future...

...measure the tension in the helical structure of spider silk, or the activity of a protein in a synapse... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...As the choice of fluorophore pairs is a key consideration for a FRET experiment, the possibilities can...PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein FRET Pairs." Sensors 16.9...

... are considered large deposits and are exceptions to our standard workflow.  Table 1: Guidelines for the...Plasmids with Addgene” blog post for a thorough guide to our online data entry process. Figure 1: Begin...application of the plasmid and will help a scientist outside your field understand if this plasmid will work...

...CRISPR Background with Our Guide Pages Other Links Check out Pawluk et al's Video Abstract Read up on All...off-target edits of base editors, which convert a nucleotide base pair to another base pair at a specific .../35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic...

...respectively. Primary cell type Gene targeted by guide Editing efficiency hiPSCs  EMX1  67% Mouse BM...in cells electroporated with RNPs using the same guide. Nanoblades also had lower off-target effects when...insertion of a DNA sequence ranging from a single nucleotide change to large insertions like the addition ...Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources Learn about viral...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ... cells that make up the human body and G-baToN provides versibility in tracking and modulating the interactions...libraries encode target sites flanked by randomized nucleotides in place of a protospacer adjacent motif (PAM... viral aliquots from our plasmid collection to provide ready-to-use viral preps. Here are some of the ...

...bigger movement in a variety of research sectors to provide open source technologies and spaces where interested...One of the aspects of this project that makes it ideal for makerspaces is the data is freely available,...projects). This allows the project to be open to a wider group of people. This is a perfect starter project...equipment becomes obsolete amazingly fast, but with a wide range of skills among our members we’ve managed ...

...powerful than it is. The nuclease domain of Cas3 is considered weak, especially compared to the power of Cas9...Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394...Liao, M., & Ke, A. (2018). Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science, 361...CRISPR-Transposons Additional resources on Addgene.org CRISPR guide Cascade-Cas3 Plasmid collection ...

...There are several facilities nationwide where you can obtain excellent quality, high titer rAAV (including...certain in vitro and in vivo applications, you can consider using unpurified AAV from the supernatant (Goodwin... in precision and variability, qPCR is the more widely used method due to the accessibility of instruments...101 series!  This post was originally written by Didem Goz Ayturk and updated by Ina Ersing in August 2023...

...of contamination for your experiments, the bright side is that you also have the power to take the added...working with so that cross-contamination can be identified. Even if some mammalian cells lines have similar...check under the light microscope will help you identify bacterial and yeast contaminants. Set up a routine...will help you avoid cross-contamination through accidental mixing and will also help you keep good records...

...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap) genes...primarily found in the culture media, you should consider performing PEG precipitation. Instead of sonicating... details. Pro tips Watch this AAV purification video! It didn’t exist when I did my first iodixanol gradient... gradient purification, but I wish it had. The video gives a brief overview of how iodixanol purifications...

...crystallographer working with Jane Richardson, provided an initial solution to this problem with the development...the surface of the protein where the nascent polypeptide emerges from the exit channel.   He then said...allow the complementary base pairing between the guide RNA and the target DNA strand, and then activate...User Resources on Addgene.org Visit Our CRISPR Guide Page CRISPR/Cas9 History Browse All CRISPR Plasmids...

...wanted to expand the toolkit. We have included a wider range of terminators than are commonly used as we...rusts and we are collecting sequences that will provide a similar resource for those involved in monocot... really and we’ll be producing an instructional video soon to make it easier for people to use the Golden...think plant biology is massively underfunded considering how important it is. I would really like to reach...

...DNA systems capable of storing data and computing inside living cells. He's also a co-founder of the BioBricks...Champion of Change, a program created as part of President Barack Obama's Winning the Future Initiative. ...proteins. That's central to everything that goes on inside a cell. Until we get a handle on precise, forward...promise that we could instead have things like peptide or RNA aptamers that act as genetically encoded...

...Microscopy. What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem....minimized through tweaks to these steps. But even under ideal polymerization and digestion conditions, loss of...Bournonville L, et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria ...Sommer C, Rizzoli SO, Danzl JG (2019) A practical guide to optimization in X10 expansion microscopy. Nat...

...inclusion of some interesting case studies, and Zimmer provides several. In Part Three of the book, called simply... and her essential-but-unlauded role in the identification of DNA’s double helix. Perhaps to the dismay...dismay of middle-aged scientists, the identification of Green Fluorescent Protein (GFP) and its development...(for funding, etc.) with that produced by the confident and flamboyant Watsons.” In Part Four, “New Science...

...less potential to become resistant to? CRISPR may provide a method for doing just that. While challenges ...problem. Above all, we need new antimicrobials, ideally ones for which the development of resistance will... antimicrobials using efficiently-delivered RNA-guided nucleases.” Nature Biotechnol. 32(11): 1141-5. ...CRISPR Plasmids for Your Research Read Our CRISPR Guide Pages Find CRISPR gRNA Design Tools ...

...numbers, but the latter eliminates the step of identifying prey proteins after screening. It is also possible...activation. Including a prey-only control also provides a baseline level of reporter activation. Vary ... And most importantly: Independently validate identified binding partners with other techniques! Minimizing... While this may be difficult to resolve for a wide range of prey proteins, there are now a number of...

... Adamson Lab recently used a CRISPRi screen to identify additional host factors impacting prime editing...subthreshold voltage fluctuations, while having high fidelity during action potentials of fast-spiking neurons... combination with miR124T targeting sequences, provides a simple yet valuable tool for microglia research...2023). Simple and Highly Specific Targeting of Resident Microglia with Adeno-Associated Virus. bioRxiv...