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...Target strand The DNA strand that base pairs with the guide RNA.   Non-target strand The DNA strand that... non-target strand is excluded as ssDNA. DSB Double-Stranded Break A cut to both strands of DNA. Indel...editing generates double-strand breaks causing deletion, transversion and translocation. Nature Cell Biology...a job for a single enzyme. Base transitions Base transversions C → TG → A A → GT → C C → G                   ...to base pair with the DNA target strand, leaving the non-target strand unpaired and accessible on the outside...binding. Pro tip! In CRISPR, the non-target strand is the DNA strand that does not base pair with the gRNA.... it the “edited strand.” Uracil excision repair leads to the first base transversion editor The story...

... are often found with cellular exosomes, small membrane vesicles or microvesicles (approx. 40 - 150 nm... diameter) that are secreted from the cellular membrane into the culture medium. AAV particles are either... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...to modify it to improve yields. For example, transfecting the producer cell line with an expression vector...proteins, nucleic acids) which possibly improve transduction properties. György and colleagues found that...that the association of AAV particles with membrane vesicles protects them from antibody recognition and...AAVs achieved so far? Exo-AAVs have improved transduction profiles in different AAV serotypes both in ...

...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: managing a team. You’ve just been...to focus on what you should be doing : writing a grant, thinking about the next project, reaching out to... transferable skills...

...learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how..., R bodies do not exert force by dynamically rearranging into different polymerization states. Instead...with proteins and remain functional, 2) break membranes outside of their natural context or 3) be modified...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...Silver, P. A. A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo. ACS Synth. Biol...

...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...

...?  Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Tranfection...define plasmid host range, identify a few of the features that confer broad host range in plasmids in nature...features to create broad host range recombinant vectors.  A plasmid’s host range is determined by the different...plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow host range, while others...identified as broad host range plasmids. The properties that influence host range are not fully understood...genes necessary for transfer initiation between bacterial hosts. Also, self-transmissible plasmids may have...examples of a narrow host range plasmid with only one ori and a broad host range plasmid with multiple oris...

...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with...cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane itself...SuperPLD hydrolyzes membrane PC to PA. A) OptoPLD is recruited to plasma membrane or lysosomes and its...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The tools...undergoes a conformational change and binds the plasma membrane under blue light CRY2-CIB1 heterodimerizes or ... separate chromosomes during mitosis, support transport of organelles and vesicles, and more. Although...demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given the long list of...

... the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template —...editing. Like other transversion editors, this editor had relatively high indel rates ranging from 4.7% to 30%...our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs...editor could excise other bases? Could other transversion editors be close at hand? Once again, this post... the end of this post! Figure 1: Base transitions are edits between the purine bases (adenine, ...bases (cytosine, thymine, and uracil). Base transversions are edits that convert a purine to a pyrimidine... Inosine excision leads to the first adenine transversion editors Uracil excision is the most common type...

...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

...your AAV packaging transfection. In my lab we regularly use the ratios below: 1 Transfer Plasmid : 1 pHelper...prevent future headaches. To help you plan PEI transfections for AAV packaging, considering using this PEI...Calculator. AAV packaging typically requires transfecting three plasmids at specific molar ratios. To ...PEI calculator for AAV packaging Use the PEI transfection calculator for AAV packaging Select the plasmid...pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue culture dish ...dish you will be performing the transfection in: 15 cm dishes OR CF5 stacks Select how many dishes or ...or stacks you plan to transfect: 1 to 10 Fill in experimental details on the spreadsheet: Name your...

...may not translate well to other cell types. To overcome this issue, try transducing with a range of dilutions...DEAE-dextran can improve transduction efficiency. In addition, this study found that transduction efficiency...passage number of a culture the lower the transfection and transduction efficiencies. Cultures should only be...others opt for a single harvest after transfecting with the transfer and packaging plasmids. A benefit of... for getting the most out of your lentiviral transduction experiments. Love your cells and they'll love...culture practices can lead to low titers and poor transductions. Cell lines should be routinely subcultured ... cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting ...

... in a nonacademic direction, there are many “transferable” skills you have developed in addition to learning...your favor? Look for opportunities to practice transferable skills in ways that will also enhance your science... concrete things you can do to develop those transferable skills while you are also learning to be an ... transferable skills...

...improve your grant writing skills. Good luck! Further advice on grant writing Since successful grant writing... during the grant application process. When my PhD advisor first asked me to write a grant application... advice on writing a grant proposal. "Murder Most Foul: How Not to Kill a Grant Application" by Vid Mohan-Ram...their advisor alone applies for these competitive grant applications; however, in some cases, your advisor...advisor may choose to allot some if not all grant duties to trusted students. For example, if the proposal...thoughts such as “I have no idea how to write a grant!”, “I don’t have time for this if he wants data ...that first experience, I learned the details of grant writing, volunteered to take the lead on various...

...terminators and resistance cassettes for selection of transgenic plants. The complete GreenGate toolkit, now available...the time required to generate multi-construct transgenic plants. In the most practical terms, what GreenGate...Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013)...

...Understanding the transition While a PhD equips you with exceptional technical skills, transitioning into management...   Strategies for a successful transition To successfully transition into a managerial role, it's essential... most pivotal decisions many PhDs face when transitioning from academia to industry is whether to stay...career trajectories within the industry, but the transition wasn’t easy - I had to reinvent myself and redefine...d like to share below. Common pitfalls in the transition (what I wish I knew before experimenting on myself...being a great scientist doesn’t automatically translate to being an effective manager. Mismanaging (no...connecting with mentors who have made similar transitions can provide you with insights, lessons from their...

...insoluble granules. We overcame this problem by engineering a plasmid which contained cold tolerant chaperones...bacteria out of their optimally suited temperature ranges and make them grow better at suboptimal ones, which...bitter cold.  Since our objective is to induce tolerance towards cold, we decided to focus on a special... of Earth’s harshest environments: corrosive pH ranges, crushing pressures, near-melting temperatures,... host cells (the simplest example would be a transformed mesophilic, for example E. coli, carrying a plasmid...hybrid system where we could incorporate cold-tolerance inducing genes within a regular mesophilic host.... And thus was born ELSA - our happy lil’ cold-tolerant E. coli! Additionally, we’re also working towards...

...repeats (ITRs) are what makes an AAV transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases...read through this challenging sequence. Transgene The transgene is the gene that will be delivered by the...Most AAV transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid ...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...(WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which ... nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...

...sybodies against membrane proteins Developing antibodies against proteins embedded in membranes is tricky because...develop antibodies against unstable targets like membrane proteins that may unfold due to camelid high body...synthetic nanobodies, called sybodies, against membrane protein targets in as little as 3 weeks (Zimmermann...lab can quickly select for sybodies targeting a membrane protein of interest using the Sybody Generation...have a concave, loop, or convex scaffold and are randomized with a defined set of different trinucleotides...serines and threonines at positions that can be randomized for creating the libraries (Zimmermann et al.... the process for selection of sybodies against membrane proteins. Image from Zimmermann et al., 2018. ...

...CRISPR-associated Transposase) by the Zhang group. Mechanism of CRISPR-transposons One of the CRISPR-transposon systems...DNA transposons have been hindered by their limited programmability: depending on the transposases involved...-guided transposition. By natively combining the DNA integration capabilities of transposases and the ...crRNA.  Three transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains... each type of CRISPR-Transposon system. (B) Mechanism of RNA-guided transposition in type I-F INTEGRATE.... Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor molecule...characteristic of the Tn7-family of transposons. Insertion of the mini-transposon can occur in two different orientations...

...third Addgene/Seeding Labs plasmid grant and plans to use the grant to order plasmids she can use as positive...in PCR-based cervical cancer screens. This third grant represents the continuation of our efforts to provide...Bengyella and Kwabena Duedu who were awarded similar grants back in June, Gladys will be using plasmids in ...will be opening the Addgene/Seeding Labs Plasmid Grant to a third round of applicants at the start of 2017...Superhero 5k Find New Cancer Resources Learn About Our Grants to Rare Disease Research Resources on Addgene.... seeding labs plasmid grant...