...so he assembled a plasmid kit designed to do exactly that. As described in the journal Science Signaling...When cancers are treated with drugs designed to hit them right where it hurts, the effects are often ...mutant complementary DNAs (cDNAs) representing core nodes that, when over-expressed, constitutively activated...activators for more cancer-related pathways and more nodes within those pathways. With support from the National...

...record breaking month and to find other posts that deserve a second look. 5 Great Apps for Lab Life This very...Stacey from Eastern Tennessee State University and provides a brief rundown of Sean’s top five smartphone ...Jessica Sacher from the University of Alberta and describes a technique you can use to find phage proteins...molecular biology tool, P1 phage. 2) The post describes an ingenious but simple technique to screen for...that, while affecting a small number of people, deserves our attention and has huge potential for the development...

... discusses some of the programs SITN runs and describes some of the more popular topics from the Science... fields as well as students from the ed school, design school, and Kennedy school to discuss the impacts...impacts of science in a social context. Episodes are driven by narrative and story telling. Chapter 5: The...The SITN Blog - Vini and Amy describe how the SITN blog is a source of scientific information for the ...underwater? Chapter 8: Work-Life Balance - Vini describes how she balances working in the lab and working...

... be spent making and testing entirely new gRNA designs. You can now easily find many validated gRNAs in...validated gRNAs? Proper target selection and gRNA design are essential for a CRISPR experiment.  The selection...headers to sort alphabetically or in ascending or descending order, in the case of numeric columns.  The data...Additional Resources on the Addgene Blog Learn How to Design Your gRNA Use the CRISPR Software Matchmaker Browse...Additional Resources on Addgene.org Browse gRNA Design Tools Read Our CRISPR Guide Pages Find CRISPR Plasmids...

...experience. Most Addgene PhD level job descriptions clearly describe that the roles do not involve bench ...submission of a job application. 3) Read job descriptions carefully Be open minded and be bold. You don...qualifications to apply for a position.  A job description is usually a wish list and the hiring manager... way to a hiring manager. Learn to confidently describe your strengths in your resume and don't be shy...

... sequence that moves around. This DNA sequence encodes the proteins the transposon needs for transposition...contain two genes: gag and pol. The pol polyprotein encodes the reverse transcriptase and integrase enzymes...ORFs) which often terminate with a poly(A). ORF2 encodes endonuclease and reverse transcriptase activities...preferred target site for integration is TA dinucleotides and it leaves behind the CAG DNA footprint from...encode reporter genes, mutagenic cassettes, or barcodes. When transposons are delivered to cells or a ...occurring Tn7 transposon found in Vibrio cholerae that encodes a Type I-F CRISPR-Cas system. This system has four...

...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation... of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed as inverted repeats... and quantify actively silencing plants. Non-destructive methods, most popularly with a fluorescent reporter...Fluorescent reporter-based RNAi constructs have been designed and widely implemented in plants, such as in the...approach, myself and others in the Luis Vidali lab designed a new RNAi method. Instead of using a fluorescent...functional APT will convert 2-FA to cytotoxic nucleotides, resulting in death. In the presence of 2-FA,...

...plasmid The packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid....com   Replication (rep) gene The rep gene encodes four proteins that are required for viral genome...cross-packaging.  Capsid (cap) gene The cap gene encodes three AAV viral capsid proteins VP1, VP2, and VP3...type tropism. Introducing changes and/or short peptides sequences into these variable loops is one method...method for changing a capsid’s tropism.  cap also encodes the Assembly-Activating Protein (AAP) and the recently...

...problem - see our recap below! Day 1: gRNA design tools: How to Design your gRNA for CRISPR Genome Editing and...given our CRISPR Plasmids and Resources a fresh new design, sorting the plasmids we have available by category...capabilities. Epigenetics: CRISPR has made it possible to design programmable epigenome editing tools, and the field...cleavage in bacteria. In mammalian systems, Cas13a degrades only the specified RNA target. Bonus: It displays...

...You’ve designed the perfect experiment – controls, conditions, and everything in between – now all you...This is where affinity tags come in – they are peptides which do not naturally exist in most expression...protein for downstream applications Below, we describe a few of the most common tags available. Maltose...antibody is an alternative to amylose resin and also provides a visual detection mechanism via applications ...especially if downstream antibody detection is desired… and yes, we have an antibody for that!   ...

...teaches you how to design a course, how to design good lesson plans, how to design test questions, and...and how to design lectures. I really built a course from the ground up.”  Science writing and editing These... was for Cactus Communications, a company that provides scientific editing services. “I was working during...

...fly fishing, and local grizzly bear attacks. He described one such recent attack as "hilarious". Welcome... Mehmet Fatih Bolukbasi from Scot Wolfe's lab described a chimeric Cas9 that is improving the nuclease... and foremost, from the custom software used to design complex genetic circuitry to the downstream output...each, and Tim Lu even compared his "memory" gene codes to a Turing machine (also discussed in the book)...also got to show off our new CRISPR page (which describes how to plan your CRISPR experiment for our new...

...smaller gel apparatus until I settled on using glass slides. With just the right amount of agarose, I could...right amount so the agarose doesn’t pour over the sides.  These tiny gels and tiny lanes required less agar...satisfying?  With practice, I got good at this despite the fact that my lab mates thought this was hilarious...minimize the required area and buffer volume.  So, I designed my own gel box (see illustration above) and bribed...plastic and solder this thing together.  One of the downsides—there was no cover so you had to be careful not...

... Antibiotic selection would only then kill the desired plants! PCR or fluorescent markers are currently... have similar drawbacks, often requiring the destruction of plants or tissue for GUS staining or are not...from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore, are useful...from these plants will be green, containing the desired mutation but lacking both RUBY and the construct...compared to other markers lends itself well to nondestructive selection. If RUBY was integrated into a CRISPR...

...our AAV preparations. We recently published a description of the VGS process and use cases in Human Gene...ligation and tagmentation preparation approaches described previously require a preliminary double stranding... the expression cassette that we want, it also includes a small amount of spurious DNA from the packaging...plasmids. We decided to use this to our advantage by designing a Python script that looks for pre-defined serotype...signature sequences in the data. Unlike previously described thermodynamics-based methods of serotype confirmation...

...technical data. These data focus on experimental design and usage of these tools, which aren’t always easy...experiment, use these questions as a guide for designing your future injection experiments. Have your data... exactly where to get that strain. Experimental design & methods: These questions are focused on injection...activate receptors? If you’re in the process of designing your experiment, read our blog post on important...to give proper attribution.  We hope that this provides a clear overview of what you need to submit data...

..., J. C., Kyrpides, N. C., Banfield, J. F., & Doudna, J. A. (2018). Programmed DNA destruction by miniature...hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps to arrive at this...largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class 1 CRISPR systems...accessory Cas proteins in the Cascade complex. Despite a tenuous grasp on how type III systems function... III-E effector was developed, called Cas7-11. Despite being in Class 1, Cas7-11 is a single protein effector...

...package. One important concept in FRET experimental design is that the targeting specificity requirements ...codon (often amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural...so small, one can insert them in virtually any desired location in a target protein, though predicted ...it all together The FRET donors and acceptors described above can be paired together to measure a wide...combinations of labeling strategies. FRET experimental design lends itself to creativity and innovation, valuable...PMCID: PMC2802762. 2. Dumas, Anaëlle, et al. "Designing logical codon reassignment–Expanding the chemistry...

...almost complete desensitization when exposed to continuous bright light. Desensitization is mediated by...discovered Marine, Anion-conducting and Intensely Desensitizing ChRs) differ from currently used ChRs in that...regulation of many genes. A number of groups have designed means of studying the effects of endogenous looping... they call the LADL Anchor. Using specifically designed gRNAs, they can then recruit these LADL Anchors...

...DNA post-cut. Using substrate DNA labeled on both sides of the break, we determined that Cas9 held tightly... single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and...approaches. Moreover, the cost of reagents is relatively modest, requiring expressed Cas9 protein, transcribed ... Blog Brush Up on Your HDR Basics Learn How to Design Your gRNA Find the Ideal CRISPR Software for Your...Plasmids for Your Research Check out Our List of gRNA Design Tools ...

...Clearly articulate the desired outcomes. Begin with the end in mind. Specify desired results. Are you delegating...expect that one person will write all the copy, design every icon, curate all of the appropriate plasmids...criteria for delegating tasks appropriately: Are the desired goals and outcomes clear? If you don’t have clear...results. What does success look like? Agree on desired outcomes. Concern yourself with what is accomplished...

...new receptors were termed Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Roth, 2016...What do smell, taste and sight have in common, besides being one of the five senses? GPCRs or G-protein-coupled...photons or odorants, to elicit downstream signaling cascades.  So let’s break down the name "G-protein coupled...then induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand...

...provide the construction plans or CAD (computer aided design) drawings of their devices, which makes it very...has allowed the community to regularly replicate designs worldwide. Distributing hardware via globally established...similar way to the biological protocol, and assembly designs based around the use of commercially available ...users outside the project to modify the raw and design files, allowing the device to take on a community-driven...of work from universities and research centers provides a potential conflict between traditional IP-based...

...instance, your Desktop directory contains the folders and files that you see on your desktop. The location...immediately write a text document with a brief description of the directory contents, at a minimum. This...for a single use, save the script as originalName_descriptionOfEdit in the same directory where it was ...Your Next Experiment Find Our List of CRISPR gRNA Design Tools ...

... to our website through the use of alt text to describe images in online spaces. Alt text is used by screen...example. Remember, our goal is to use words to describe the image in a way that helps visually impaired... your alt text, use all of this information to describe the image helpfully. You and your readers are ...learned a lot from listening to others' alt text descriptions, and we’d love to hear yours as well.  More ...write alt text for any image you’re struggling to describe in the results. And many journals accept alt text...

...Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated from different..., and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which...Nishimasu et al., 2018). The David Liu Lab similarly described xCas9, which displayed the ability to target multiple... also high off-target activity, which may be undesirable in many applications. At the time of our first...post on choosing a Cas9, the Feng Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed...

...first generation of CRISPR-Cas9 plasmids were not designed with gene activation in mind. “We had not managed...Konermann and her colleagues in Feng Zhang’s lab describe just such a system, which they call CRISPR/Cas9...from the structure to guide and improve activator design,” she said. “Before it was a little like tapping...instead of at the protein’s C terminus. With that new design in place, Konermann immediately saw an order of...consistency and validation with >80% effectively enriched guides per gene hit, and 100% validation of the top 10...

...element. This link provides a more extensive list of yeast auxotrophic markers and includes the associated...differ from the bacterial elements previously described. Why do scientists use yeast vectors? Yeast are... that come with working with prokaryotes. This includes but is not limited to: rapid growth, ease of replica...most commonly used selection markers in yeast and provides the element needed to overcome the auxotrophy ...

...been common in fields like math and physics for decades (see: arXiv.org), but have been only adopted by...course, preprints don’t replace journals and other modes of scientific communication. Researchers often post...to receive deposits of plasmids that have been described in a preprint. In fact, over 1,500 plasmids in...Depositing plasmids that were used in a preprint provides all the same benefits as sharing those plasmids...resources on the Addgene blog Summer SciComm Series: Modes of Communication Intro to Our Summer SciComm Series...

...different labs have recently published work that provides a solution to this problem. A miniature CRISPR-Cas...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...Nature Biotechnology paper showcases an improved design of the guide RNA that caps the 3’ end with a structured...that mismatch repair (MMR) interfered with the desired resolution of prime editing intermediates. Building...sequence. As with other prime editing tools, the desired edits are templated by the 3′ extension of the ...

...protein-coupled receptor, one of the Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Armbruster...bottom). The images show examples of dendrites (blue shades) in the monkey amygdala. Red and green arrows point...deciding on the appropriate tag, the experimental design should be the main guidance. The results from our...Raper J, Hu X, Paré J, Bonaventura J, Richie CT, Michaelides M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH...

...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable...Additional resources on the Addgene Blog  How to design your CRISPR gRNA CRISPR 101: Ribonucleoprotein ...

...of the Retroviridae or Retrovirus family, which includes the gamma-retrovirus and lentivirus genera, have...dividing and non-dividing cells. More detailed descriptions of the lentiviral elements and the 2nd and 3rd...#48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is ...building block Entry vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial...single ecotropic or amphotropic MuLE lentivirus provides significant genetic power and allows high throughput...

...widely used in neuroscience. In this post, I’ll describe how rabies virus (RABV) can be used in the brain... by modifying the wild-type RABV genome, which encodes a single envelope protein called rabies glycoprotein... neurons integrated into the brain over time (Deshpande et al., 2013). The study found that at early time...Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, Lepier ...Pubmed PMID 26190977. 6. Osakada F, Callaway EM. Design and generation of recombinant rabies virus vectors...

...introducing a desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One...potential for treatment is no longer an accurate descriptor; progress in clinical therapy is more fitting...One treatment currently in clinical trials includes Cas3 (the shredder member of the Cas family) as part...CRISPR therapies have made significant clinical strides since we first wrote this blog post. The future...

...Plasmids designed to express genes in a given host cell type are generally broken down into two broad...post-transcriptional additional of multiple adenine (A) nucleotides to the tail of a messenger RNA transcript. The...structure of the hairpin-DNA complex is thought to destabilize the transcription complex, initiating cleavage...transcription of the template and initiating the desired cleavage event, although some engineered terminators...downstream elements. A high affinity terminator may be desired for multi-cistronic constructs where high termination...

... asking the panelists not to use any slides for the event. Slides are for presentations, not discussions...discussions. I will rarely work with a panel if slides are allowed. Don't put your panelists behind a table. ...organizations, I asked audience members to stand and describe unique nonprofit models that differed from the...around to meet and greet audience members. Having dessert, drinks and/or coffee available only after the ...

...addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique...polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents and a series of heating...protocol page, protocol video and reference on how to design PCR primers.  Types of PCR Since the invention ...  Digital droplet PCR or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration...Polymerase Chain Reaction (PCR) Protocol: How to Design a primer  Molecular Biology Reference Molecular...

...library, each plasmid encodes the mutated spike protein and a barcode, and is designed so that the SARS-CoV....1074/jbc.RA120.01289     Novel protein cage provides simple enzyme protection in yeast bioproduction...enzymes in a reaction pathway, the MPyV system provides a simple, efficient method for protecting a single...- Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by anhydrous...

...fluorescent proteins, it’s important to note that despite their distinction by name, chromoproteins are actually...chromoproteins have a shorter history. In 1987, scientists described an unusual blue-colored protein from the jellyfish... chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and ...recombination events: The plasmid carrying the desired genetic modification integrates into the genome...rhamnose-inducible promoter. The resolution step leads to the desired mutation and excision of the vector backbone. To...

...Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain... 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 effector, type III with a Cas10...CRISPR transposase systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR ...maximum efficiency of the process. Various labs have designed all-in-one Cascade plasmids — such as the Tomoji...

...(Are the inserts of different sizes?), and the desired coverage for the library (How important is it that...  The most important factor to consider is your desired library coverage, since this will help decide what...similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can...should always experimentally test the protocol you design to ensure that it works for your library before...Addgene. If you have any specific questions about designing your library amplification protocol, feel free...

...append their proteins of interest with unrelated peptides that already had high quality antibodies available...existence of high quality antibodies for well defined peptides. Munro and Pelham’s first published peptide tagging... tumorigenesis.  Using two different synthetic peptides corresponding to linear sequences within the middle...that perfect. One of the known limitations was described by Schüchner, et al., who published a paper showing...works and allow users to better troubleshoot and design experiments. It turns out that even with the most...

...such a system is likely to introduce additional nucleotides flanking the insertion. If the DNA ends anneal...MMEJ may also introduce, substitute, or delete nucleotides in addition to the expected deletion, but the...Building on the lab’s previous work, Sakuma et al. describe a detailed protocol for MMEJ-mediated knock-in...PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA The gRNA should...binding in mammalian genomes as assessed by CRISPR design tools at crispr.mit.edu. They tested their system...

...fluorescent output. The GEVI family of gadgets includes Ace, JEDI, and many more. They can be targeted...component as well as a small molecule which we will describe in more detail below. Types of Voltage Indicators...Drosophila to phosphatases from sea squirts. This design structurally separates the two components of the...membrane, a concern with several of the previously described systems. Several adaptations of this method have...voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or both. Targeting these systems...

.... Individual gRNAs are designed in silico using publicly available gRNA design software and synthesized... be independently validated to ensure that the desired modification produces the phenotype you screened...in the first place. With the proper experimental design and validation practices, CRISPR libraries can ...CRISPR Plasmids Read our CRISPR Guide Find tools to design your gRNA ...

...human therapeutics (e.g. using Sleeping Beauty). Despite their diverse utility, DNA transposons have been...(V. cholerae INTEGRATE - hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components...the same promoter, and a donor plasmid (pDonor) encodes the mini-transposon. pEffector and pDonor can also...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...even very large genetic cargo, at one or multiple desired genomic loci. Many applications of these systems...

...individual molecules. Poor photon budgets are a widespread issue with fluorescent proteins—even the thriftiest...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical... the complexity of the system. A particularly desirable property for small-molecule labeling strategies...size, brightness, and cell permeability. In past decades, further refinements to this established dye structure...spectral range. Nevertheless, these fluorophores were designed as antibody and oligonucleotide labels for fixed-cell...

...beta-lactam resistant population to the antibiotic, despite this resistance gene being encoded by a high-copy...currently undergoing a renaissance. Yosef et al. designed a two-phage CRISPR system to resensitize antibiotic-resistant...immune system, may someday be used to target the hardest-to-kill bacteria. In using phage-derived vectors...Ahmed A. Gomaa, and Rodolphe Barrangou. “A CRISPR design for next-generation antimicrobials.” Genome Biol...Research Read Our CRISPR Guide Pages Find CRISPR gRNA Design Tools ...

...DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first...editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s...named ABEmax. In 2020, two papers were published describing additional ABEs evolved from ABE7.10 that have...cytosine and adenine base editing, called CABE-Ts and TadDEs without additional fused components (Lam et al....editors have a narrow editing window of only a few nucleotides, so they are tightly constrained by PAM availability...