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...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...

...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...

... with reduced error rate for long sequence knock-ins Visual barcodes for live cell clonal multiplexing... with reduced error rate for long sequence knock-ins by: Lucie Wilson Le Cong’s lab has developed a method... large inserts. However, this system has a much lower error rate. For a one kilobase insertion, the researchers...limitations of number of flourescent proteins that can be inserted into a cell without disrupting its functions...peptides with four different fluorescent proteins, and inserting each visual barcode into its own subclone...TurboID-nanobody protein and allows TurboID to start labeling proteins in close proximity to the GFP-tagged protein (...need to generate “single-use” transgenic lines. Instead, researchers can generate and maintain transgenic...

...are appropriate for generating knockouts or knock-ins, or introducing mutations and tags, while a catalytically...homology directed repair (HDR) template to create knock-ins, point mutations, or to add a tag, you will also ...used in conjunction with activator or repressor domains to control gene expression. Base editors can help...will evaluate the outcome: Will you be detecting insertions/deletions using a mismatch repair assay? Or is... to identify and enrich positive cells. Likely contains a selection marker to generate stable cell lines... (packaging limit ~4.5kb). CRISPR elements are inserted into an AAV transfer vector and used to generate...Topic Page Learn about CRISPR Delivery Using Ribonucleoproteins (RNPs) Read Tips for a 1st Time CRISPR User...

...amplification. Given the inefficiency of HDR for knock-ins, some labs have used NHEJ for whole plasmid integration...is thought to be ideal for HDR-independent knock-ins, but this possibility is still being explored. More... DSB repair. NHEJ joins the ends of a DSB in an error-prone fashion, with insertions and deletions common...construct is inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-... of PITCh-based genomic insertion would compare to CRISPR sticky-end insertion using the nuclease Cpf1...introduce additional nucleotides flanking the insertion. If the DNA ends anneal incorrectly, MMEJ may ...constructed with 5’ and 3’ microhomology to the insertion locus flanking the GFP-Puro cassette. Three double...

...concept and go beyond the basics - diving into the ins and outs, tackling complex questions, and giving ...around the corner. Here at Addgene, we’re always inspired by the new seasons, so we've made some big summer...Social Media Are you following us on Twitter or Instagram (or Facebook or LinkedIn) but want to hear and...love sunshine and warm weather - check out our Instagram all summer long to see what kind of shenanigans...

...true protein behavior. Traditional epitope knock-ins maintain the native protein context, but they still... answer research questions before creating knock-ins.  Figure 1: Schematic of HiBiT tagging technology... or unique surface epitopes for many proteins, but some proteins do not have such epitopes. These classical...Studying proteins in their natural context is one of the biggest challenges in biology. From tumor suppressors... factors, some of the most clinically-relevant proteins are also the hardest to study. One common strategy...luminescent signal, strong enough to detect from proteins expressed at their endogenous levels. HiBiT is...interference with the native function of tagged proteins and facilitates precise CRISPR-based tagging, ...

...CRISPR can also be used to generate cell line knock-ins of patient mutations which are variants of unknown...drugging some targets – the dreaded ‘undruggable’ proteins – having multiple hits or an entire pathway of...

...be used in a lab? Here we’ll be talking about the ins and outs of this classification system, including...minute. Rinse the slide briefly with tap water. Flood the slide with Gram's iodine solution. Rinse briefly...immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide ...specific procedural instructions, including timing, preparing the smear, and mixing the stains and solutions...biologist named Christian Gram realized that certain stains were retained only in some species of bacteria....negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com/photos/occbio/7092385559...laboratory setting. When designing a plasmid to be inserted into a specific bacterium, it can be important...

...needs. For instance, I had one colleague who preferred detailed instructions and regular check-ins, which ... made similar transitions can provide you with insights, lessons from their experiences, and practical...

...One-on-Ones: Managers should set up regular check-ins with their employees. 15-30 minutes weekly or monthly...who could potentially be impacted by a project. Instead, invite the experts on the topic at hand. Invite... people of an updated protocol), send an email instead. The 3 A's - Agendas, Action items, and Accountability...parts where people had questions or suggestions, instead of reading the whole thing together. How long...FInd Tips on Picking a Research Project Read Other Inside Addgene Posts Resources on the Addgene Website...

...screen vs. a selection and we are here to discuss the ins and outs of both approaches. Screens and selections...players in a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that ...specifically looking at cells that can survive. Instead, all manipulations are assessed, regardless of ...considering. RNAi screens are similar to CRISPR, but instead of knocking out the genes of interest (or the whole...

...developed new CRISPR-based methods for various knock-ins, discussed in our CRISPR 101 eBook. It’s hard to...targeting vector designed to insert a resistance gene. The vector contains a neomycin resistance gene (...sequence using a specially designed vector that contains sequence homology to the locus of interest. To...negative selection marker HSV-tk is used to select against random recombinants.   Design your targeting...recombination, the negative selection marker selects against improper, random recombination into a different...After “floxing” the resistance gene through the insertion of flanking LoxP sequences, you can remove the...Figure 2: A knockout targeting vector designed to insert and later remove a resistance gene. In this construct...

...ligands (Glypicans and Integrins), components in the synapse (Neurexins and Neuroligins), and common epitope...synaptic cleft proteins are currently under development. Figure 2: Key adhesion proteins and receptor–ligand...Recommended Applications PI Return to top Integrins Integrins are a family of mechanotransduction receptors...and secreted proteins from a collaboration between Addgene and IPI. Antibodies... Antibodies Institute for Protein Innovation... Innovation Antibody Collection Institute for Protein Innovation Antibody Collection Axon Guidance Antibodies...Plasmids (Link opens in a new window) Welcome to the Institute for Protein Innovation (IPI) Collection! Here,...

... Plasmid Collections Fluorescent Proteins FRET Fluorescent Proteins: FRET FRET Pairs FRET Calibration...conformational changes. Organic dyes, fluorescent proteins, and other molecules have been demonstrated for...Pairs This table lists some popular fluorescent proteins used as FRET pairs along with their properties...calibration standards consisting of donor and acceptor proteins fused with linkers of varying lengths. Description...Description Article ECFP-EYFP FRET standards, fusion proteins with linkers of varying length Evers et al. (2006...understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers mCerulean...mTurquoise2-mVenus FRET calibration standards and fusion proteins Feldmann et al. (2023) Protocol for deriving proximity...

...Signaling Strains Browse Addgene's collection of synthetic biology related bacterial strains. SynBio Bacterial...Strains for use in Synthetic Biology. Plasmid... Plasmid Collections Synthetic Biology Bacterial Strains Synthetic...Synthetic Biology: Bacterial Strains SynBio Resources SynBio Home Browse All SynBio Assembly Standards SynBio... Bacterial Strains Search the table by keyword or sort by the table headings. Click on the publication... this depositor's lab. Plasmid Description Gene/Insert Antibiotic Resistance PI Publication Back to Top...

...Collections Brzezinski Lab CRISPR Collection Brzezinski Lab CRISPR Collection The Brzezinski lab investigates...empty backbones that utilize Golden Gate cloning to insert the desired guide RNA sequence by digestion of ...utilizing one of the dual guide shuttle plasmids to insert a second guide within a U6-guide cassette. This...Cloning Protocol (PDF, 237 KB) Dual guide cloning: Brzezinski Lab Dual Guide Protocol (PDF, 990 KB) Plasmid...R., Abraham, J., Hensley, A., Jones, K. L., & Brzezinski, J. A. (2021). Initiation of Otx2 expression ...Goodson, N. B., Kaufman, M. A., Park, K. U., & Brzezinski, J. A. (2020). Simultaneous deletion of Prdm1...

...homology directed repair to make precise edits or knock-ins. Editing efficiency decreases drastically once your...away from the insertion site, so HDR will be difficult if there’s no PAM near your insertion site. Base ...upstream of the PAM. Even if there’s a PAM near your insertion site, the corresponding gRNA might be undesirable...multiple PAMs should have a fitness advantage over proteins with single PAM recognition. Created with...were capable of cleavage, they tested the clones against an NNN PAM library in bacteria where cleavage leads...

...Allen Institute for Cell Science has deposited plasmids to generate fluorescently tagged proteins of key...Depositor Collections Allen Institute for Cell Science Plasmid Collection Allen Institute for Cell Science Plasmid...Subcellular Localization Plasmids Protocols The Allen Institute for Cell Science has produced the first publicly... will be able to generate fluorescently tagged proteins of key cellular structures in human cell lines...additional cell line information from the Allen Institute for Cell Science data portal (Link opens in a ...cellular structures. Addgene ID Plasmid Allen Institute ID Tag Protein Structure 87420 PXN-EGFP AICSDP...114406 SMC1A-mEGFP AICSDP-57 mEGFP SMC protein 1A Cohesins 114407 NUP153-mEGFP AICSDP-50 mEGFP Nucleoporin...

... from Allen Institute's Genetic Tools Atlas (Link opens in a new window) . Allen Institute for Brain Science...Enhancer Plasmids The Allen Institute for Brain Science Enhancer Collection contains best-in-class AAV plasmids...cell populations were tested in mouse brains. This collection contains plasmids for cell populations in the...created by the Allen Institute for Brain Science. Depositor... Depositor Collections Allen Institute for Brain Science...Science AAV Enhancer Collection Allen Institute for Brain Science AAV Enhancer Collection Plasmids Publications...drive cell type-specific expression. The Allen Institute for Brain Sciences has screened thousands of enhancers...

... your best self to the training experience.  The ins and outs of being a trainee There is a lot of advice...technical mentorship from a ‘bench mentor’. In some instances, the bench mentor is also the scientific mentor...different, so let your mentor know what yours is! For instance, some people like to ask questions throughout ... clarification at first, but in the end everyone wins. Your mentor learns where you are at and the level...

...their controls!). This blog post will discuss the ins and outs of controls for biological experiments, ... experimental (?) groups were run on a western against a protein standard (P) to check for tau molecular...antibody specificity is to test the primary antibody against wild-type and knockout samples. In this test, wild-type... each experimental run as long as the protocol remains the same.    Figure 2: Microscopy-based ...positively charged groups with generally sticky proteins such as bovine serum albumin. Secondary antibodies...all secondary antibodies are included. If, for instance, you omitted a mouse IgG2a isotype primary antibody... cells only (for example, propidium iodide only stains dead cells). Compensation controls address the ...

..., the efficiency of Easi-CRISPR for simple knock-ins and floxed models varies from 8.5-100% of pups born...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.” Genome Biol. ...standard laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria....remain more difficult to make. For example, large insertions, like a fluorescent protein knock-in, suffer ...intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand breaks ...other undesired outcomes, including one loxP site insertion and one NHEJ-repaired indel, often occur. ...your donor ssDNA. Here, we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et...

...of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used...standard cloning (where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will ...vector and insert DNA to use. Combine the following in a PCR or Eppendorf tube: Vector DNA Insert DNA Ligase...transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector ratio is usually sufficient...construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly...join the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and ...called "sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends are...

...choose enzymes that: Flank your insert, but do not cut within your insert Are in the desired location in...commonly used. Follow the manufacturer’s instructions. Isolate your insert and vector by gel purification Run...DNA . Ligate your insert into your vector Conduct a DNA Ligation to fuse your insert to your recipient...plasmid to insert. It is also important to set up negative controls in parallel. For instance, a ligation...cut elsewhere on the plasmid Will result in your insert being in the correct orientation in the recipient...of your gene!) Are in frame with tags or fusion proteins in the recipient plasmid (if you are creating ...specifically designed test digest later to verify that the insert was cloned in the correct orientation. If you cannot...

...the gel can indicate if the plasmid contains the expected size insert. By selecting the appropriate enzyme...manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert and ...plasmid. Verifying Insert Orientation by Restriction Digest If you clone an insert into a vector by a ...orientation of the insert. By choosing an enzyme (or enzymes) that cuts within the insert, but significantly...diagnostic digest to verify plasmid size, verify insert orientation, and more. Protocols...restrictions sites. Often, the size of the plasmid insert and vector backbone are known and thus this technique...the entire construct or excise some or all of an insert from it. Before beginning your diagnostic digest...

...commonly used. Follow the manufacturer’s instructions. Isolate Your Insert and Vector by Gel Purification Run...DNA . Ligate Your Insert Into Your Vector Conduct a DNA Ligation to fuse your insert to your recipient...plasmid to insert. It is also important to set up negative controls in parallel. For instance, a ligation... to choose enzymes that: Do not cut within your insert. Are in the desired location in your recipient ...our cDNA into the recipient plasmid. Remember to insert your DNA in the correct orientation in the recipient... Purify the PCR Product Run PCR to amplify your insert DNA. It is important to use a high fidelity taq...of DNA). Once you have cut out and purified your insert and vector bands away from the gel, it is important...

...white precipitate will be formed which contains the bacterial proteins and genomic DNA. Incubate tube on ice...tube for 5 min at 12,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles...min at 4°C. Notes: Pellet contains the precipitated DNA. Supernatant contains residues, salts, and water...If you will be using a kit, follow the kit's instructions. If you want to perform plasmid purification... This step brings the pH back down causing the proteins and genomic DNA to precipitate, while leaving ... smaller plasmids free in solution. Pellet the proteins and genomic DNA by centrifugation, and remove ...that has been purified away from the bacterial proteins and genomic DNA. Depending on the method used,...

...your institution’s Bio-Safety office to receive permission and institution-specific instructions. You ...bacteria D. Screening for Inserts D.1 Recommended materials D.2 Screening for inserts E. Producing Lentiviral...Consortium has built a library of shRNAs directed against 15,000 human and 15,000 mouse genes. Addgene is...selection. Figure 1 : Map of pLKO.1 containing an shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb... Negative control vector containing non-hairpin insert. pLKO.1 – scramble shRNA Negative control vector...targets for your gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection...Additional design rules can be found at the Broad Institute's TRC shRNA Design Process . To minimize degradation...

...Appropriate restriction enzyme (see manufacturer's instructions for proper ammount) Approrpriate restriction...restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips Procedure...an appropriate reaction buffer by reading the instructions for your enzyme. Pro-Tip If you are conducting...for 1 hour. Always follow the manufacturer’s instructions. Pro-Tip Depending on the application and the... the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours ... involve incubating the reaction at 70 °C for 15 mins, or purifying the DNA via a purification kit, such... cleanup kit . See the enzyme manufacturer's instructions for more details. To visualize the results of...

...competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock...many genetic modifications to create bacterial strains that can be more easily transformed and that will...efficiency, we recommend that you follow the instructions that came with your competent cells. Pro-Tips...out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate...be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. ... the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing ...bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will...