...the light they emit. Proteins that give off light include chemiluminescent proteins, like luciferases, ...Nobel Prize for fluorescent proteins The importance of fluorescent proteins was recognized in 2008 when... discovery of GFP The story of how fluorescent proteins like GFP came to be used as research tools is ...Using these samples, he was able to isolate two proteins from the jellyfish’s photoorgans; the first, which...and shift its emission spectrum. New fluorescent proteins with new colors Around the same time, researcher...vibrantly colored. He hypothesized that GFP-like proteins could be responsible and began studying these ... fell into place, as they, taken alongside the insights provided by the studies in Tsien lab, covered ...

...FP-chimeric proteins. Photoactivatable fluorescent proteins (PA-FPs) are fluorescent proteins that display...Fluorescent proteins (FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate...Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral (protonated) and...irreversible PA-FPs are the photoconvertible fluorescent proteins (PC-FPs). PC-FPs can convert from one fluorescent...Selected Irreversible Photoactivatable Fluorescent Proteins Protein Oligomeric State Activating Light ...Selected Reversible Photoactivatable Fluorescent Proteins Protein Oligomeric State Activating Light ...actually harm your cells before your experiment even begins! If you are performing multicolor experiments, ...

... like chromoproteins, fluorescent proteins, and luminescent proteins. While fluorescent proteins and luminescence...fluorescent proteins? Before going into the difference between chromoproteins and fluorescent proteins, it’s...whereas chromoproteins do not fluoresce at all or fluoresce at very low levels. Chromoproteins in nature... history of fluorescent proteins in molecular biology research, chromoproteins have a shorter history....do these organisms have chromoproteins? One reason may be that chromoproteins protect corals from solar... Advantages of using chromoproteins for molecular biology Because chromoproteins absorb visible light ...substrate, chromoproteins don’t require other substrates for screening. Thus chromoproteins can be used...

...generating sybodies against membrane proteins Developing antibodies against proteins embedded in membranes...and Y-shaped because it contains two large heavy chains and two small light chains that stick out to form...trying to develop antibodies against unstable targets like membrane proteins that may unfold due to camelid...the process for selection of sybodies against membrane proteins. Image from Zimmermann et al., 2018. ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https:...identified nanobodies that bind to coronavirus spike proteins (Wrapp et al., 2020), which sit on the surface...special camelid antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  ...

...fluorescent proteins. Additional fluorescent protein tools Photoactivable fluorescent proteins (proteins that... Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed...danger. In this post we’ll focus on how fluorescent proteins can and have been used to monitor cellular movements...specimen. It allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated...that have been tagged with different fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation...Cellular trafficking studies enabled by fluorescent proteins The optimization of GFP and its derivatives by...changed everything. For the first time, fluorescent proteins gave researchers the ability to track immune cells...

...applications of fluorescent proteins. These fusion proteins enable the observation of proteins in living cells and...successfully in countless fusion proteins. Choose monomeric proteins and avoid a sticky situation Next...oligomerization of fluorescent proteins is a concern. The propensity of fluorescent proteins to oligomerize is known...requirement is that the site of insertion does not interact with other proteins or biomolecules. Examples of...create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition...blog Read our fluorescent proteins blog posts Download the fluorescent proteins 101 eBook Read our plasmids...they like, the design and engineering of fusion proteins is relatively straightforward. However, generating...

...AnltWfKuQg pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020 Contest 2: Deck ... to #DeckTheLab with virus snowflakes from @CVRHutchinson #SciArt #STEAM #WinterWonderlab @Addgene Make...picture or video for either contest on Twitter or Instagram by December 31, 2021. Let us know which contest...and the winners will be revealed on Twitter and Instagram shortly after. Have fun, be creative, and enjoy...celebrates and decorates, follow us on Twitter and Instagram as we share pictures of our decked out spaces....

...Luciferase Can Be Used In Concert with Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter...reporters are generally less bright than fluorescent proteins, but have the advantage of lower background levels...

...acceptor proteins is not optimal, FRET may not occur or be detected even if the two proteins form a complex...Circular permutation and receptor insertion within green fluorescent proteins." Proceedings of the National...Imagine being able to determine whether two proteins are within 10 nanometers of each other, or measure...with fluorescent molecules, including fluorescent proteins, has led to the alternative acronym Fluorescence...microscopy. Don't FRET - Use these FRET resources instead! As the choice of fluorophore pairs is a key consideration...overwhelming given the large assortment of fluorescent proteins available. Fortunately, several excellent reviews...Many empty vectors containing these fluorescent proteins are available on our FRET Resource page and can...

... fluorescent HPLC (proteins or other molecules) Isolate or quantify cells, proteins or other molecules...created by Roger Tsien’s lab contain two fluorescent proteins (a FRET pair) separated by CaM and M13. When calcium...increases FRET transfer between the fluorescent proteins. FRET is highly amenable to biosensor creation... experimental setup? Consider what kind of instrumentation you have available, and the conditions of your... fluorescent proteins...

...its unbound state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore...flexibility is their broad range of targets, including proteins, peptides, amino acids, drugs, metal ions, and...One of the advantages of oligonucleotides over proteins is their modularity, which is lost as soon as ...paved the way to new biosensor possibilities. For instance, further selection of Spinach with a target metabolite...applications that lend themselves to aptamer-based instead of protein-based fluorophores, including: Environments...Environments with higher temperatures, since proteins can denature while aptamers are more stable and can...Nucleotides, in general, are far less immunogenic than proteins. Assays that require recognition of ions or ...

...Backstrom, a biochemist in the Vanderbilt Eye Institute and Tonia Rex's lab. A common strategy to determine...Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized on a reduced glutathione...protein. However, oligomerization of biotinylated proteins on avidin can be controlled easier than formation...biotinylation selectivity A common strategy to biotinylate proteins involves amine-reactive biotin derivatives that...incorporation and to a lesser extent, the number of added biotins per molecule cannot be well-controlled. The lack...and HisTag, which were “buried” in a few of our proteins that lacked TEV. Although we were unable to cleave...we tested, it may be possible with other fusion proteins - but it would remove both the AviTag and HisTag...

...background. In many instances, variations of the product’s specific application instructions or “optimized ...technical support specialist for 4 years. Read on for insights from my time in technical support including tips...individual stories and get (sometimes refreshing) insights into how other scientists think and work. Most... other roles in industry. Many thanks to Eric Perkins and Stephanie Hays for images used in this post...

...aeruginosa. These toxins were a mix of Type VI secreted effector toxins, toxin-antitoxin type toxins, and phage...Bacterial toxins for counter-selection In 2015, the Chen lab harnessed a group of bacterial toxins to facilitate...cassette with one of three toxins from the Khetrapal paper. We selected the three toxins so that at least one...sacB-based allelic exchange vector. This vector contains sacB, an chloramphenicol resistance cassette, ...phage holins. They hypothesized that genes that had evolved specifically for toxicity would allow stronger...i.e. sacB). Importantly, to prevent activation of toxins before the second crossover step, they tested their...enabled very efficient recombineering.     We were inspired by this work and sought to develop these negative...

...reproducibility, and community-building. The transparency begins with sharing sequence information for the antibodies...community demand. Figure 1: Antibody against PSD-95 from the Trimmer Lab. Information about ... represent the official views of the National Institutes of Health....

... plant, grow, and harvest grains every time you want to bake a cake. Instead, you go to a store and purchase...start with writing demo details, step-by-step instructions on how to demonstrate the feature. For example...

...Plasmid Sharing Or read more about what happens Inside Addgene   ...

...Even before fluorescent proteins (FPs) came into wide use, there were a variety of ways to monitor cell...cell, organelle, and protein localization. For instance, you might dye your cells and look at them under...hybridization experiments. In many cases fluorescent proteins have usurped old methods or complemented them ..., either in whole cells or as a pool of fusion proteins localized to a specific region of the cell. For...problematic aggregation-prone tetrameric fluorescent proteins can be. Cytotoxicity, improper localization, and...from previously developed monomeric fluorescent proteins mKO and mCherry, respectively. While Tsuboi et...blue-red maturation times (Table 1). These separate proteins should be useful for monitoring cellular events...

...purification using qPCR or digital droplet PCR. Insect cells Insect cells are a less common option for the generation... viruses that specifically infect insects. This is what makes insect cells a popular choice for protein...your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in cell lysis,...Mammalian versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses...different cell types, including plants, mammals, and insects. In this post, we will compare two of the most ...most widely used systems, mammalian and insect cells. Whether you’re new to in-house virus production or ...passage (transfection). This differs from typical insect cell preparations, which we talk about below. Processing...

...magnetoresponsive proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic...(among others, flavins are very effective) and to see if any other fluorescent proteins display a similar...fluorescent proteins respond to magnetic fields?” and “What are magnetoresponsive fluorescent proteins good ...deposited their plasmid with Addgene, hoping to inspire others to explore the phenomenon, improve the tool...and the interactions of organic molecules like proteins with magnetic fields generally appears to be weak...that described magnetic responses of cryptochrome proteins or organic fluorophores (Karki et al., 2021; Lee... to search for a magnetic response among other proteins that might serve as a starting point for developing...

... at Albert Einstein College of Medicine. Several sets of near-infrared fluorescent proteins (NIR FPs) ...Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. ...Biliverdin chromophore Near-infrared fluorescent proteins (NIR FPs) were derived from natural photoreceptors...mouse. Image courtesy of Axel Nimmerjahn (Salk Institute for Biological Studies) Multiple uses: From...blue-light controlled optogenetics, such as channelrhodopsins and tools based on light-oxygen-voltage (LOV...Daria Shcherbakova is a faculty member at Albert Einstein College of Medicine. Her expertise is development...development and applications of fluorescent proteins, genetically encoded biosensors, and optogenetic tools....

...career skills for positions both inside and outside academia. Inspiring future scientists At Addgene, we...

... of toxic or insoluble proteins Origami2 (DE3)** Streptomycin and Tetracycline Contains highly active...that are insoluble, act on DNA, or are enzymatically active. For this reason, recombinant proteins are typically...most expression strains to accommodate high protein levels including:  ompT: Strains harboring this mutation...proteolysis of the expressed recombinant proteins. lon protease: Strains where this is completely deleted (designated...reduce proteolysis of the expressed proteins. hsdSB (rB- mB-): These strains have an inactivated native restriction...Expression Systems Table 1: E. coli expression strains  Note: All strains are derived from the E. coli B strain...DE3) (RifR) Expression of unstable proteins Tuner (DE3)   Contains mutated lac permease whch allows for...

...strain. Common E. coli strains used in the lab Most of the commercial strains you find today are marketed...changes found in E. coli strains. Table 1: Common gene mutations found in E.coli strains Gene(s) Description...glutamine insertion supF (tyrT)   Suppression of the amber (UAG) stop codon by tyrosine insertion λ-thi...of the popular strains, their genotypes, and their primary use in the lab. These strains are all based ...plasmids with R6Kγ origin; contains a mutant allele of the pir gene that maintains the plasmids at ~250 copies...optimal promoter for your gene of interest, painstakingly cloned into the perfect empty backbone, made...hard to count the number of different commercial strains of E. coli currently available – a quick Google...

...PUF proteins! A newly available PUF Assembly Kit makes it possible to devise RNA binding proteins to hit...applying the Golden Gate cloning method to human proteins known as Pumilio/fem-3 mRNA binding factors (PUF...step, researchers can now assemble designer PUF domains for RNA specificity engineering. “The RNA binding...University of Illinois at Urbana-Champaign. The PUF proteins include eight modular repeats, each of which binds...explained, the specificity of other RNA binding domains depends on the secondary structure of RNA. As a...eukaryotic RNA.” Endless possibilities with PUF proteins The tool she and her colleagues led by Zhao described...repressor. In principle, however, designer PUF proteins can be fused to other components to lend them ...

...which other proteins it forms complexes with. These “colocalization” studies give insights about the proximity...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...its role in a disease, researchers often isolate proteins of interest and examine them using biochemical...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...You can take advantage of the many Fluorescent Proteins (FP) available for microscopy, by cloning your...final destination. As an example, most secreted proteins are produced in the endoplasmid reticulum, modified...extracellular destination. At the end of their life cycle, proteins may be transported through the vesicles to the...

...about these proteins is that they are monomeric fluorescent proteins. Fluorescent proteins that oligomerize...University, Indiana University, and the Karolinska Institute developed mNeonGreen via 21 substitutions to LanYFP...fluorescent protein mNeonGreen. This fluorescent protein joins mTFP1 and mWasabi, as fluorophores from Allele Biotechnology...deposited at Addgene. What makes these fluorescent proteins unique and what can they be used for? Let’s take...Nathan Shaner and colleagues from The Scintillon Institute, Allele Biotechnology, Florida State University... sequence identity to other common fluorescent proteins and therefore can be used in immunoprecipitation...immunoprecipitation experiments with other fluorescent proteins. Find mNeonGreen plasmids here! mTFP1: a monomeric teal...

...deliver channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one...conductance compared to current channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain...engineering channelrhodopsins There are three main challenges to engineering channelrhodopsins to have improved...membrane localization of transmembrane proteins, like rhodopsins, are highly constrained and poorly understood...understood. It’s hard to design new channelrhodopsins with high-throughput proteins engineering methods, like directed... than existing channelrhodopsins CoChR and/or ChR2(H134R). Designer channelrhodopsins paired with a systemic...expression of light-gated ion channels such as channelrhodopsins (ChRs) makes them light-responsive. When light...

...Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses. Feng S et ...measure the signal produced when the candidate proteins’ interaction brings the pieces of the FP back ... to help!  General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments...canonical beta-barrel structure. Most frequently, the proteins are split between strand 10 and 11 to yield large... with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R,...(1-10) and mNG2(11) Improved split fluorescent proteins for endogenous protein labeling. Feng et al. Nat...

...binding proteins waiting to be explored and exploited. With better characterization, these proteins could... sequence the inserted DNA and characterize your new putative host cell-binding proteins! Many thanks...their bacterial prey. Currently, receptor binding proteins (RBPs, the host recognition factors of phages)...constitute one of the most popular classes of phage proteins to study. These are highly useful for the biotech...Addgene's MIcrobiology Pages The problem: Host binding proteins are hard to find Sequencing and cloning phage ...that allows for a phage genome to be probed for proteins with host-binding activity without the need for...genome that encode for functional cell-binding proteins in the (all-too-common) event that homology-based...

...fluorescent proteins requires some thought. The first step in choosing fluorescent proteins for your multi-color...aware of what fluorescent proteins are available. With new fluorescent proteins being published every month...fluorescent proteins, I maintain an interactive graph and table of the best fluorescent proteins currently...Choosing compatible fluorescent proteins To choose a set of fluorescent proteins to be imaged together, you...between the two fluorescent proteins. If you must image fluorescent proteins whose spectra overlap, there...fluorescent proteins, but these are beyond the scope of this post. Are your fluorescent proteins compatible...wavelength red fluorescent proteins like TagRFP or mRuby2 than longer wavelength proteins like mCherry. For background...

...fluorescent proteins found on the red side of the spectrum are not GFP derivatives, but are instead related... of use, GFP and other fluorescent proteins have become a mainstay in molecular biology. Scientists can...study the interactions between two proteins or between two domains of a protein that undergoes conformational...Bioluminescence and fluorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed...aqueous solvent. This tightly-packed structure explains the importance of the entire GFP protein, which...engineered. Table 1 lists a few common fluorescent proteins and their mutations relative to wildtype GFP. ...red-fluorescent protein repertoire; however, these proteins are unique from GFP and the mutation definitions...

...case when proteins undergo post-translational modification with fatty acyl chains. For instance, myristoylation...available green fluorescent proteins emit light. The pKa of fluorescent proteins can be accurately determined...2017). GFP can only be used for tagging proteins GFP fusion proteins are made by connecting its DNA code ...by using protein domains that specifically bind the molecule of interest. For instance, pleckstrin homology...bright red fluorescent proteins (e.g. mScarlet) or infrared fluorescent proteins (Chernov et al., 2017)...GFP Read our Fluorescent Proteins Blog Posts Download the Fluorescent Proteins 101 eBook Resources on ...microscope, and finally (iv) good toleration in fusion proteins. Since GFP is such a well-validated, all-round...

...ReFigures are published instantly and are editable by the author at any time. Insights contained in your ReFigure...researchers, we wondered how we could make the insights gained during this time more visible and thereby... ReFigure saves time, knowledge and makes your insights discoverable. Watch this quick video to learn ... ground again and again as they don’t save the insights they glean from a reading session. To remember...ReFigure making it easy for others to discover your insights on a topic. Beyond collating other’s data, you...field, while providing you with visibility. For instance, long before a study with multiple patients was...which can convince reviewers of your expertise and insight. We recommend sharing ReFigures on your Linkedin...

...Addgenie Eric Perkins presented a poster a few years ago at a CRISPR meeting.Read his Instagram feature here... what lies ahead. Thus the “Meet an Addgenie” Instagram interview series was born. Over the six months... Addgene’s Director of Product Management Eric Perkins, who has been in many roles in Addgene since he... really get me where I haven’t gotten myself.” Perkins noted that there is a more emphasis today about...Shannon Rinaldi in the Addgene lab! Read their Instagram profiles here (Kenkel, Rinaldi)! But leaving...Vedantam with our resident dinosaur! Read her Instagram feature here. One of our QC Scientists, Shreya...Read all of the Meet an Addgenies features on Instagram! Staying true to your values While going through...

...immunohistochemistry methods, these proteins are routinely fused to ‘tag’ or ‘label’ proteins to facilitate its detection... that fluorescent proteins might not always be the best option. Fluorescent proteins can interfere with... on the Addgene blog: Fluorescent Proteins 101: GFP Fusion Proteins – Making the Right Connection.   ...activity of neurons and other brain cells. Since the opsins or chemogenetic receptors used in these techniques...detection. Fluorescent proteins are most widely used tags because they emit endogenous fluorescence that... vivo, or post-mortem experiments. Fluorescent proteins can also be identified using antibodies and immunohistochemistry...immunohistochemistry techniques. Thus, these proteins provide a versatile method to identify the expression...

...from Joachim Goedhart: Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection.  mEmerald...Updated May 10, 2021. In the world of fluorescent proteins and their use for imaging cell biology, Michael...impressive 1,350 scientists from more than 275 institutions all over the world. In the course of all those...plasmid backbones spanning a rainbow of fluorescent proteins “It’s a great collection with over 300 backbones...interest isn’t in the ORF collection, the fluorescent proteins are, which makes mixing and matching a relatively...exponentially as far as new targets and fluorescent proteins are concerned.” For more information on choosing...targets since the Davidson lab always tagged new proteins with mEmerald first, Baird says. From F-Actin ...

...behaviour in social insects, obviously as all people are,” says Susan. Her passion for insects lead her to start... Keown, a staff writer at the non-profit Fred Hutchinson Cancer Research Center. Susan Keown’s science...science writing internship with the National Eye Institute (NEI), where she wrote about NEI research for ...applied and I got it.” Science writing at Fred Hutchinson Cancer Research Center Work as a Science Writer...Addgene Other Resources The National Cancer Institute (NCI) has an internship similar to Susan’s NEI...

...of a transgene construct containing fluorescent proteins (XFP) of different colors to label an organ or... a combination of XFP are expressed. Each cell remains the same color for its entire lifetime and daughter... label neurons. Genes encoding the fluorescent proteins are organized in tandem with a single promoter..., stability, and brightness of the fluorescent proteins used in the system. One such system is Brainbow... because by default, they are not fluorescent. Instead, they incorporate a truncated, non-fluorescent ...a unique combination of all of the fluorescent proteins from the injected construct. The fluorescent protein... protein expressed in a single cell maintains its color over the entire length of the cell, even axons...

...conducted in Professor Cepko's lab and to get some insight into her management and mentoring styles. If you'd...Cepko's recent work on developing GFP-activated proteins, read our recent blog post. Check out the break...al. “A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation...

...lacked an “off-switch” for Cas proteins. Discovery of anti-CRISPR proteins That is, until the discovery...of anti-CRISPR proteins in 2012 (Bondy-Denomy et al., 2012). Anti-CRISPR (Acr) proteins are phage-derived... found here. How do anti-CRISPR proteins work? Anti-CRISPRs proteins are highly diverse but most block... be as effective. This is where Acr proteins come in. Acr proteins are small enough that they could be...technology is constantly evolving. Variants of Cas proteins can be used for genome editing, activating gene...s activity. The concern is that the longer Cas remains active in a cell, the greater chances there are...CRISPR-Cas immune system of bacteria. These Acr proteins were originally found in type I CRISPR-Cas systems...

...current vaccines against the variants? Alejandro Balazs, a Group Leader at the Ragon Institute of MGH, MIT...vaccines against SARS-CoV-2 variants, this study was the first to compare so many different strains. It was...study. Figure 1: Neutralization levels against spike pseudovirus decreases depending on the variant..., Tegally H, Lustig G, Giandhari J, Pillay S, Wilkinson E, Naidoo Y, Karim F, Ganga Y, Khan K, Bernstein...

... plant hormones called auxins to conditionally and reversibly degrade proteins of interest fused to a ...   New toolkit for N or C terminal tagging of proteins for degradation using AID Article contributed ...ubiquitin ligase pathway needed for AID. By tagging proteins with a 7kD degron called mAID and expressing the...developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116 cells with mAID. Using this method, ...generate conditional alleles of a few essential proteins (Natsume et al., 2016). Now, the Kanemaki lab ... plasmids for the N- or C- terminal tagging of proteins with mAID and other tags (Yesbolatova et al., ...sufficiently suppresses basal degradation of tagged proteins, a common problem of the AID technology (Yesbolatova...

...into the 2in1 vector instead of the GatewayTM cloning BP reaction, where insertion of the GOI occurs through...technique using small scaffold proteins to identify novel binding proteins that bind to either the light... anti-CRISPR podcast segment Anti-CRISPR (Acr) proteins potently inhibit type II CRISPR systems including...temporally ubiquitous, making them a rather blunt instrument for controlling CRISPR. To bring greater control...optogenetics. Through the use of light-sensitive protein domains, they have developed a system to control CRISPR-Cas9...Bubeck et al., 2018). They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into...study the electrical activity in the brain by inserting electrodes into the brain. But this process is...

...majority of knock-ins were occurring 24 hours post-injection, making screening for knock-ins much more efficient...unc-119) (Dickinson et al., 2013). These approaches allowed effective recovery of knock-ins, but did result...visible phenotype enriches for knock-outs and knock-ins at other genomic loci. The first approach – co-CRISPR...representative co-selection experiment, five knock-ins were recovered from seven rescued F1 animals (gel...from 11-100% of F1 animals carrying precise knock-ins, and homozygous knock-in animals can be obtained ...repair templates are critical for efficient knock-ins (Ghanta et al., 2020). Together, these improvements..., obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence considerations...

...donor vector to create knock-ins of fluorescent proteins, fusion proteins, or transcriptional regulators...research! This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli...knockout libraries for cancer research Creating knock-ins with GeneWeld vectors GeneWeld from Jeffrey Essner...library from David Bikard’s lab. The Bikard library contains ~93,000 unique gRNAs that target random positions...

...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base pairs or bigger... to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with only a few base...appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-in. We’ll also...edit – is it any frameshift mutation? A specific insertion or deletion? A knock-in? A base edit? Once you...interest will get the job done. In other words, any insertion or deletion that isn’t a multiple of three will...simple algorithm to quantify editing efficiency by insertion and deletion frequency. Fig. 1: Overview...

...get done. However, this is not the case for knock-ins. Knock-in mutations typically require an exact DNA...for HR-related mutations before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of...Banan. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. J Biotechnol., 16(4): 387-390...less traveled and generating a knock-in cell line instead of a knock-out? We’ve got you covered in this blog...template. The highest HDR efficiency is observed when inserts are within 10 bps of the break, making the cut ...further, you can inhibit cell cycle regulatory proteins responsible for the transition out of S phase,...events compete with NHEJ-mediated edits of small insertions and deletions.   Start your knock-in experiment...

...selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background. Their new method uses ...added to the plates to select for candidate knock-ins. These candidate animals can then be further screened...it possible fluorescently label proteins in C. elegans, they inserted the SEC into a synthetic intron ...sequences are inserted into the genome. Figure 1: Self-excising cassette schematic from Dickinson et al...Self-Excising Drug Selection Cassette. Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. Genetics. 2015...Homology directed repair (HDR), which enables insertion of custom genomic modifications, is very robust...making the detection of rare events difficult. Dickinson et al. sought to create a new and efficient selection-based...