...see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity...intended application — here, flow cytometry — is the desired target, using one or more of the approaches outlined...interpreting your data therefore necessitates careful design of RNAi sequences, confirmation of RNA knockdown...the target at different levels, it is possible to design experiments where the performance of the antibody...We find that cell tracker dyes are helpful in designing such panels, so that pre-stained cell lines can...pass type-1 membrane protein), which has been designated CD365 in the most recent HLDA workshop (HLDA10...recognized by either antibody was not shared. Tables describe that the antibodies both recognized the target...

...the virus doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1,... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target the 5’ long terminal ...prevent reinfection of those cells. Kaminski et al. designed gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed...could be used to combat this problem, just as ART includes multiple drugs to lower the odds of developing...strategy may be a better option than direct cleavage. Despite the potential difficulties in translating these...

...dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and they must...wasting leading to death at around 30 years of age. Despite the amount of research conducted on DMD, there ... over time, stem cell editing would be highly desirable. Should stem cell editing occur, the CRISPR components... for Duchenne muscular dystrophy. gRNAs can be designed to flank a single or multiple exons that, when...Short-term CRISPR expression would be especially desirable, as it would reduce the potential of off-target...require robust stem cell editing to maintain the desired phenotype. Precision genome editing faces the challenges...Covello G, Denti MA. Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases...

...plasmid with its elements described below.   Vector Element Description Origin of Replication ...molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. A plasmid is a...why each is important. Our “Plasmids 101” series designed to educate all levels of scientists and plasmid...basics and spend more time developing cleverly designed experiments and innovative solutions necessary...utilized in the lab are usually artificial and designed to introduce foreign DNA into another cell. Minimally...the lab. Addgene’s Molecular Biology Reference includes information about molecular cloning, how to choose...

...Image Search results includes images that can’t be reused without attribution, despite filtering for images...Initially I didn’t have access to professional-grade design software, like Adobe Illustrator, and I didn’t ...Googling and some trial and error, I found some free design software that let me create graphics that better...of Adobe Illustrator. There’s both a web and a desktop version of this software. Vectr has layers, which... this post. It’s open source and available for desktop use for Windows, Mac OS X, and Linux. Inkscape ...The web-based collection of over 20,000 icons is designed by professional medical illustrators with input...

... CRISPR plasmid tools and numerous experimental design decisions to make, it makes sense that scientists...editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment Q1: Should...comparable efficiency to the optimized chimeric design but with better accuracy (in other words, lower...nickase system is based on the Cas9 D10A nickase described in Figure 4 of the Cong, et. al, 2013 Science ...from the Zhang group. Learn more here. Q2: When designing oligos for cloning my target sequence into a backbone...reference.  More FAQs, CRISPR protocols, and gRNA design tools Need more questions answered about CRISPRs...CRISPR plasmids at Addgene Find protocols and gRNA design tools: List of CRISPR protocols developed by a...

...multiplexibility in various species and describe an assortment of applications. Despite the ubiquity of CRISPR/Cas ... store a record of invading phages but to also destroy the phage upon re-exposure (2, 3). More specifically...complex binds to the foreign DNA, a cut is made to destroy the invader. Wikipedia, accessed 25 November...engineering. The diversity of the CRISPR/Cas systems provides powerful lines of defense against invading phages...CRISPR/Cas inspired genome editing was initially described by Cong et al. and Mali et al. in January 2013...Nine months later, more than 1500 publications describe work to improve the tool’s specificity, orthogonality...

...assign four different barcodes to each of the gRNAs and to embed these barcodes within the gRNA sequences...lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized GFP ...method that makes use of guide RNAs with internal barcodes (iBARs) in their loop region. After infecting ...bioRxiv 2019. https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast...intracellular Ca2+ (bottom panels) equipped with destabilized GFP calcineurin reporter (blue) and stained ...

... researcher in Jacob Corn’s lab, describes how you can better design your donor DNA template to achieve...Detect Zika Virus The RNA-based toehold switches described in this post are engineered to bind RNA sequences...Boston University School of Medicine, this post describes a tool you can use to study basic virus biology...researcher in the Silver Lab at Harvard University, describes R-bodies, “force generating protein machines that... Connie Cepko’s Lab at Harvard Medical School describes how AAVs function and indicates important things...from the Jackson Laboratory for Genomic Medicine described Casilio, a system that uses modular RNA binding...

..., creative writing, designing and curating exhibitions, animation, graphic design, and more. I recall ... career in neuroscience—whatever that entails. Despite being engrossed by the theory and findings of brain... my curiosity did not ultimately translate to a desire to do the research itself. Several years of exclusively...more creative faculties into motion, such that a desire to once again write for fun eventually saw an English...experimental aspect of my thesis: a survey instrument designed to measure how individuals respond to issues of...SciComm Series: Cognitive LoadSummer SciComm Series: Modes of CommunicationIntro to Our Summer SciComm Series...

...for Gibson assembly. The steps following primer design and the PCR process itself are very similar to ...the downstream steps. For this example, we will describe how to copy a cDNA from one vector into a new ...YGOI) for ligation into a recipient plasmid. Designing primers for PCR based cloning     The basic...: Do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement...

...fluoresce: FlipCherry. FlipCherry provides a proof of concept that the designed used in FlipGFP can be applied...range and high brightness. The Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which ...prevent binding, the β strands in FlipGFP were redesigned so that they are parallel to each other and thus...fluorogenic reporter toolkit, the Shu Lab also designed a red fluorogenic protease reporter based on superfolder...PMCID: PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for...Central PMCID: PMC5026494. Zhang, Qiang, et al. "Designing a green fluorogenic protease reporter by flipping...

... C-terminal dronpa fusions. With these various designs, Xin has been able to develop photoswitchable Intersectin... other researchers will be able to use similar designs to create a wide variety of enzymes and proteins...post. Fatima Enam: Biosensors for human milk oligosaccharides Fatima Enam is currently a graduate student...fluorescent protein-based biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important...nonspecific in the second. Fatima therefore set out to design and produce two biosensors that could quickly and... and reliably detect HMOs. Fatima’s first design is a whole cell biosensor with two components - an enzyme...recently presented her work at her home town in Bangladesh where it was well received. In addition to her...

...up walls of text. Since our guides are heavily used, our graphic designer made most of the Antibody Guide... quickly." Final design (right). Immediate and immense regret of the graphic designer not shown.   ...blog, website, social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to ...communication between our content team and our graphic designer to make sure things are clear, accurate, and in...Reference image (left) provided to our graphic designer upon request that the science communicator "just...

... encoding multiple guides. While two guides require two arrays for Cas9, two guides can be streamlined...applications. This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag ...target multiple genes within a single cassette provides increased simplicity for combinatorial genetic...cost-effective alternative to commercial ladders which also provides a useful yet low-pressure teaching opportunity...less than half the size of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy...

...connecting each genotype with vulnerabilities) to design a therapeutic strategy that works. But, those darn...information on each cancer clinical sequencing report includes rare variants that haven’t been characterized?...based on maps of protein structure. Sometimes this provides a great deal of clarity. But, often these methods...signatures or cancer phenotypes. These assays were designed to complement the one assay per gene approach ...27478040. The lung cancer mutant collection is described in this paper. Systematic Functional Interrogation...the genetic pathway reference collections are described in this paper Systematic morphological profiling...  The genetic pathway reference collection is described in this publication We also wrote an extensive...

... Blog Learn Best Practices for Designing Your gRNA Read How Barcodes Can Be Used in Lineage Tracing Read...genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome...their functions. The continual advancements in gRNA design necessary to achieve maximal on-target and minimal...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that...quantitatively analyzed by sequencing its set of barcodes. CombiGEM is highly flexible and can accommodate...PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to maximize activity and minimize off-target effects...

...sometimes have to design new primers to sequence the important features. You can use primer design software such...such as Primer3 to help you design your custom primers. Remember, when designing a sequencing primer, you... to differ between plasmids, and selecting or designing a primer specifically to target this region, as...instructions on other factors to consider when designing your primers can be found here. Finally, you will... to Verify Your Plasmid Learn How Addgene Uses Barcodes to Track Samples Resources on the Addgene Website...

...engineering, with the ultimate objective of genome design.” It’s genetic engineering on a genomic scale, ...Systems Biology in November, Voigt and Alex Nielsen describe what’s possible when multi-input CRISPR/Cas genetic...with many guide RNAs connected because the signal degrades at each layer. If we could fix these things, then...many deposits to Addgene over the years. Can you describe the toolbox you are putting together?  Voigt: ...projects. Not from the ground up, but so that genetic designs of that scale could be created. Someone could take...doing is developing software to allow people to design systems of this scale. That will be coming out ...next 100 years, we are really going to be able to design new materials and chemicals as sophisticated as...

...application. It typically includes overview information, key dates, funding description, research plan, award... setbacks, and potential alternative routes, Provides opportunities to convince others why a project...aspects are understood. While reading the funding description, pay attention to the keywords used and think...grant applications restrict the space available to describe the proposed work. Thus, every single sentence...literature and offer educated insight into what the desirable features of future technologies should offer in...project are clearly stated and align with the description of the RFA and funding institution. Emphasizing...

...spreadsheet Data Field Description Plasmid Name Use a descriptive plasmid name and match the...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose... in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options ...does this plasmid do?” in <200 characters. A descriptive purpose focuses on the practical application ... Primary Vector Type The vector type should describe how your plasmid is intended to be used. Choose...

...Resonance Energy Transfer (FRET) was originally described by Theodor Förster in 1948 as a variation of the... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...FRET probe, try searching PubMed for articles describing the FRET tool that you are looking for and check...created the sensor that you need. FRET biosensors designed to measure specific small biomolecules or gene...biosensor, Cameleon (Miyawaki et al., 1997), was designed to measure intracellular calcium and published...1997. Since that time, numerous advances in probe design, fluorescent proteins and microscopy equipment ...molecules, detect cellular processes and signaling cascades, quantify mechanical tension (a molecular “spring...

...editing landscape. The need for PAM-less editing   Guides for CRISPR are typically selected by the ‘quality...sequence is suitable, but there is no PAM next to the desired cut site, then a further-away gRNA where there ... may wonder: how close is close enough for the desired edit to still occur if the cut site is sub-optimal...it depends, but generally the efficiency of the desired edit is reduced by over 50% when the cut site is...sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented equally, that’s only a 1/16 chance... an ideal site: moving several bps away from a desired site does not have the same devastating impact ...Between SpRY and several of the other Cas variants described, nearly all genomic landscapes should be editable...

...Have no fear, Addgene is here to help!  General Design Figure 1: Two proteins are fused to the ...from the associated paper:   Color Base FP Description Article PI Blue EBFP2 EBFP2(1-10) and Capri...contains both fragments of split mVenus (aa 155), includes I152L in N-terminal fragment An enhanced recombinant... Dan Mulvihill mVenus Split mVenus (aa 155), includes I152L in N-terminal fragment Constant rate of ...reversible complementation. TagBiFC (split HaloTag) provides maximum flexibility in color labeling with exogenous...single-molecule tracking in live cells.  Controls  When designing experiments, it’s important to include proper ...post for more on BiFC principles and experimental design. We hope you find these plasmids useful. Good luck...

...cassette via Cre recombination. Each knockout includes unique barcodes and universal priming sites to ensure ... the Chi-Bin Chien lab (Kwan et al., 2007) and includes four promoters, six fluorophores with nonoverlapping...The set of CyRFP constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression...editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated with...codons) with kanamycin cassettes and were carefully designed to allow for easy removal of the resistance cassette...essential in B. subtilis 168. Each individual strain includes a xylose-inducible dCas9 paired with a single,...across a variety of plant species. This toolkit includes TALEN and CRISPR nucleases, nickases, and gene...

...BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite protein in the...favorite protein in your hand? At 3D Molecular Designs, we create physical models of proteins using 3D...off a small, family-run business – 3D Molecular Designs – to commercialize some of the best instructional...reaction to this model on his blog. Or, for a description of how effective this model was in explaining... up close and personal, visit the 3D Molecular Designs web site. Or, if you are visiting a local classroom...check out our hands-on teaching tools that are designed to make the molecular world real for students....BioMolecular Modeling and the CEO of 3-D Molecular Designs.     Additional Resources on the Addgene Blog ...

...unstructured templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were...Delivery to Intended REcipient Cells Through Envelope Design). This modular approach combines an interchangeable...Cell type-specific delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org...LOV-Turbo plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes...that had been trafficked from the “source” to “destination”. Images adapted from Lee and Cheah et al. 2023...Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4 protein ticker tape recording. B) HEK ...bioRxiv, under CC-BY-NC 4.0 license. This system provides a unique new way to record and quantify the dynamics...

...than plasmids without this DNA. They eventually designated this fragment as the ccdB region (for “coupled... serves as a counterselectable marker in the destination vector. Successful cloning occurs when the ccdB...restriction endonucleases in plasmid addition was first described by studies of EcoRI and Bsp6I in Escherichia coli...systems. As with the other toxin-antitoxin systems described above, one part of the pair is more stable than...toxin-antitoxin system discovered. Unlike the systems just described, the hok/sok systems consist of three components...long-term experiments. They’ve also helped researchers design and optimize cloning methodologies using counterselection...Central PMCID: PMC177048. Thisted, Thomas, and Kenn Gerdes. "Mechanism of post-segregational killing by the...

... screen them for your desired edit. Good news! Remember the primers you designed to generate your pCAG-EGXXFP...CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase...that do it for you. One such option is the CRISPR Design tool supplied by the Zhang lab. This simple website... and cons of using many different types of gRNA design software. When you are happy with your selection...Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko et al., 2014. Not all...Resources on Addgene.org Find Software Tools to Help Design You gRNA Find CRISPR Plasmids for Use in Mammalian...

...optogenetics than neuronal activity. Researchers have designed a variety of photoswitchable elements into many... and limitations – for example, microtubule-destabilizing drugs are impossible to target to specific subcellular...plasmids if you want to make a stable cell line. This design allows highly localized, rapid and reversible microtubule...Rho GTPase activity. A) Schematic of Opto-RhoGEF design and activity. B) OptoTIAM (magenta) expressed in...processes in cells and tissues. And, this Opto-RhoGEF design could be extended to other GTPases. The Chow Lab...plasma membrane. In previous publications, they described tools to control formation of lamellipodia and...Jaber, Z., Gardner, K. H., & Chow, B. Y. (2022). Designing Single-Component Optogenetic Membrane Recruitment...

...your desired edit are all you need to generate a knock-in.   General considerations for designing a repair..., and ultraviolet light. In this post, we will describe the mechanism of homology directed repair, one...ssDNA templates (referred to as ssODNs (oligodeoxynucleotides)) are commonly used for smaller modifications...increase the frequency of edits, researchers have designed self-cleaving plasmids that liberate the targeting...information on HDR and CRISPR. Our CRISPR 101 series was designed to help explain the basic principles driving CRISPRs...et al. PLoS ONE 9(8); (2014). PubMed. Making designer mutants in model organisms. Peng, Y. et al. Development...

... your cells based on their size, the cytometer provides you with two measurements: forward scatter (FSC...SSC-H, or FSC-W/SSC-W, respectively. H, A, and W describe the shape of a histogram, which graphically represents... of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W results from...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell...depends on cell type and laser voltage. The voltage describes an electric potential that can be applied to the...comparing two markers simultaneously, we commonly describe the resulting populations as double positive (...that there are three parameters — H, W, and A — describing the intensity and duration of the detected signal...

...any DNA sequence in the way that they like, the design and engineering of fusion proteins is relatively...fusion proteins and highlight some aspects of their design.  Protein size and shape matters The green fluorescent...necessary for post-translational modifications. Despite these potential issues, GFP has been used successfully...consist of random amino acid sequences or (ii) designed to form an inert, unstructured peptide and therefore...C-terminus of the fluorescent protein. I prefer to describe the different parts of the fusion from N- to C-terminus...2011).  Although structural information may guide design, it is advisable to generate multiple constructs...over-expression artifacts The introduction of DNA that encodes a fusion protein adds protein to the existing pool...

...optimized. How should I design my mutagenic primers? The key is to design primers that are the same ...generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure...PCR that uses mutagenic primers to introduce all desired codon changes (step 1 in the image above). A second...ssDNA is packaged into phage and then extracted. Besides taking a couple days to generate the phage-derived... The one-pot saturation mutagenesis technique described in Wrenbeck et al is a PCR-based approach for ...Treating the plasmid with ExoIII and ExoI then fully degrades the nicked strand. Synthesize mutant strand: A...

... While it is certainly possible to select for desirable traits using traditional plant breeding approaches...modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already been used...wheat, mushroom, and rice (Khatodia et al., 2016). Despite the almost universal functionality of the CRISPR...genes in plants using the same general experimental design principles developed in other model organisms (...nanostructures (Zhang et al., 2019) or cell-penetrating peptides (Santana et al., 2020) and plants viruses (barley... through Addgene. In addition to the plasmids described above, Addgene carries several useful CRISPR toolkits...CRISPR Multiplexing Tools Validate Your Genome Edit Design Your gRNA Additional Resources on Addgene.org ...

...comment reflected the desire for a flexible generic solution, rather than an ELN designed for a specific research...to insufficient and non-standard record keeping despite scientist’s best efforts. We all have software ...practices, their opinions towards ELNs, and their desired functionalities i.e. main priorities of different...was a significant barrier to ELN adoption. This includes financial outlay, staff hours, troubleshooting... to require on-going maintenance and support.” Besides that, academic institutions tend to think about...required for regulatory purposes in many labs. Besides this being of crucial importance when applying ...

...comments section.  FX cloning kit This kit is designed to be used with Type IIS restriction enzymes and...this kit have been used to purify a variety of peptides including antibiotic efflux pumps and ankyrin ...et al. "A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo...followed by leader sequences (encoding small leader peptides) downstream of which you can clone your gene of...Shine-Delgarno sequences used in this so-called bicistronic design (BCD). The 49 vectors in this kit can be used to...(2015): 31-37. pCri system The Gomis-Ruth lab designed the pCri system to simplify the protein purification...for cloning. Furthermore, some plasmids contain peptides to facilitate protein folding and/or protease ...

... Roth BL (2015) DREADDs (Designer Receptors Exclusively Activated by Designer Drugs): Chemogenetic Tools...less invasive and more flexible as it relies on designer ligands that can be simply introduced by injection...lab and Takafumi Minamimoto’s lab known as deschloroclozapine (DCZ) has been developed to meet these requirements...should be carefully controlled for in experimental design.   Optogenetics Chemogenetics Timing Fast ...Find our chemogenetics and optogenetics science guides ...

...CRISPR inhibition (CRISPRi). A) Lentiviral vector design. B) Strategy for leak-free transcriptional control...Organoid EasyTag workflow combines thoughtfully designed plasmid components with Fluorescence Activated... heavy optimization. Claudia Vickers’s lab has designed novel SL fluorescent biosensors by integrating... rather than on the fluorescence intensity, it provides a direct and quantitative detection of SLs. The...biosensors could have a major economic impact.   Design and mechanism of action of the receptor-based SL...targets all mouse genes with 10 gRNAs per gene. The guides are optimized to maximize on-target cleavage and...

...controls and be able to use this information to design thoughtfully and thoroughly controlled experiments...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the...the Fc, allowing you to detect this undesirable interaction. When non-specific interactions are suspected...nonspecifically with positively charged groups, such as aldehydes in fixed samples. To address this, block positively... will be a useful starting point for you when designing rigorous experiments. While this blog touched ...