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We narrowed to 957 results for: Cre

Showing: 281 - 300 of 957 results

  1. Design Tips for Prime Editing

    Type
    Blog Post
    ... edited. Created with BioRender.com. Add silent mutations near point mutations to create 3-base (...target can increase the odds of incorrect editing outcomes, especially when MMR is inhibited. Created with ...edit. Figure 1: Prime Editing schematic. Created with BioRender.com. The pegRNA specifies both...PBS) and reverse transcriptase template (RTT). Created with BioRender.com. The first base of the ...to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. What to edit The sequence... mismatched bases can often evade MMR (right). Created with BioRender.com. For specific prime editing...nicking sgRNAs ~50 bp from the prime editing nick. Created with BioRender.com. PE3b and PE5b: In this...

  2. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...activation. Created with BioRender.com. Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-...targeted demethylation is associated with increased transcription. Created with BioRender.com. Tet1 initiates...the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified...This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation and activate transcription. A lentiviral...DNA oxidation and repair factors that allow for increased gene activation at the targeted site compared ... of transduced cells. Grant Challen’s lab also created constitutive (pCMV-dCas9-D3A) and Tet-dependent...is associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1...

  3. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest...sequence preferences must largely be ignored. Created with BioRender.com. The hammer: Gene knockout...terminus of the protein, to maximize the chances of creating a non-functional allele. For a 1 kilobase gene...primary importance (more on this below). Created with BioRender.com. The jigsaw: Editing by ...the need for single cell cloning and subsequent screening for successful edits. This is a very time consuming...constrained by the desired location of edit; efficiency decreases dramatically when the cut site is >30nt from the...options that can greatly expand gRNA options. Created with BioRender.com. Two newer technologies ...

  4. Transferable Skills: Negotiation

    Type
    Blog Post
    ...closer to an innovative resolution. Creative negotiation can also decrease or entirely eliminate the feeling...and everyone leaves unhappy.   Negotiation as a creative problem-solving process Although at first glance...negotiating, what if we instead approached it as a creative problem-solving exercise? Asking questions like...involved and be used as a guide to point you toward a creative solution where everyone wins. What are the underlying...but still be beneficial to both.  Coming up with creative solutions where everyone wins can initially feel...person. It can especially be difficult to think creatively when you are in the middle of a frustrating situation...practice will make negotiating feel more natural. Creative negotiation skills will help you strengthen relationships...

  5. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ...of themselves. This increases the number of copies of the TE while also increasing the size of their host...mutagenesis screens that detect loss-of-function or gain-of-function mutations. In these screens, the transposons...transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters and enhancers...Then they can be inserted into new target sites. Created in BioRender.com.   Class II TEs: DNA transposons...which produces a target site duplication (TSD). Created in BioRender.com.   Autonomous vs. non-autonomous...capacity >100 kB, although integration efficiency decreases with cargo size. Sleeping Beauty has a close-to-random...can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo capacity of ~...

  6. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...during your in vivo work is to screen you gRNA in vitro first. In vitro screening is a simple and fast method...Researchers are now using this system in ever more creative ways, (Wang et al., 2013, Cho et al., 2014). There...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers to the...same time, and choose the most effective ones to increase your chances of gene modification in vivo. Find...embryos to develop. Once they are born, it’s time to screen them for your desired edit. Good news! Remember...

  7. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...compose the secretory pathway, endocytic vesicles, mitochondria, lysosomes or they are secreted into the ...your cloning strategy and decide whether you will create an N- or C-terminal fusion based on your experimental...residues form inappropriate disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs ...and a sequence intended to localize them to the secretory pathway, a dark pool that is mislocalized to the...misfolded, blocked from proceeding through the secretory pathway, and retained in the ER. FPs have been...recent publication illustrated the quantitative increase in fluorescent signal achieved when utilizing ...optimized moxFP as opposed to standard FPs. The increase in signal is not due to a brighter FP, but rather...

  8. Building and Validating Recombinant Antibodies: A Case Study

    Type
    Blog Post
    ...the process that was used to create this important collection.  Creating the Plasmids To begin constructing...were able to greatly increase the number of functional constructs that were created. This greatly limited...construction and validation of some of these tools created by the Trimmer Lab that will be the first antibodies...that had already been thoroughly validated. The creativity in employing a variety of cloning techniques ...from cryopreserved hybridomas, immortalized cells created by fusing an activated B cell that produces the...VH) chain sequences. Fusion PCR was performed to create an amplicon that  was cloned into a plasmid backbone...plasmid to express the target antigen could be screened in a high-throughput manner. Cells were labeled...

  9. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    ...you can be incredibly frustrating. Clarifying the words —> decreasing frustration —> increasing the happiness...in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was relatively... an engineer that you want the LIMS software to create a barcode for your plate, you will get in trouble...happening. Our solution for this problem was the creation of an Addgene lexicon. The base lexicon was and...received formal definitions in our lexicon. We created documents and presentations, gave real life examples... field or technology in a consistent way. Just creating a lexicon was not enough. We also had to enforce...the terminology of a new field. Annotate: When creating important documentation, including SOPs or project-related...

  10. Your Top Requested Plasmid in 2015!

    Type
    Blog Post
    ...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If...metalloproteinase-9 secretion, and identifying lysine methyltransferase substrates. We’re incredibly happy that...interest to mammalian cells. It was designed to have increased lentiviral titer as compared to previous lentiviral... population of cells as part of pooled library screens (1,2). The human and mouse GeCKO libraries, targeting...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...you get full library coverage in pooled library screens. Availability and Use LentiCRISPR v2 is available...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87...

  11. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    ...NG-Cas9 (PAM: NG) to create NG-ABE8.xm/d and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With....xm/d showed a 1.6x median increase and Sa-ABE8.x-m/d a 2x median increase in editing frequency over ABE7.10...combination with a synthetic library of TadA sequences to create the eighth generation of adenine base editors, ...selection. As a result, 8 mutations in TadA could create all three edits and were thus more efficient. Informed...behavior to ABE8.x-d. Overall, ABE8s showed a median increase of 1.94x editing activity compared to ABE7.10....ABE8.17-m/d, ABE8.20-m/d) to evaluate further.  The creation of accidental insertions and deletions (indel)...ABE7.10 can be a problem. Therefore, the authors created ABE8 constructs using a catalytically “dead” S....

  12. A Look at Addgene's QC Process

    Type
    Blog Post
    ...where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender.com....effort into our quality control (QC) processes to screen for and reduce these errors. These processes allow...  Figure 2. Addgene’s basic QC process. Created with BioRender.com. Once Addgene receives deposited...process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching...issue? At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the...decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene...resolve. Sequencing and assembly issues Sequence discrepancies are not always a result of mutations in the ...

  13. Plasmids 101: Colony PCR

    Type
    Blog Post
    ... type of primer pair is also great for screening clones created with the same backbone but that contain...it's in the plasmid Can be used to screen clones that were created with the same backbone because primers...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...is, allowing you to use the same primer pair to screen for the presence of many different inserts. The...one primer anneals to the insert. A simple way to create this type of primer pair is to mix-and-match insert-specific...test your colony PCR primers before using them to screen colonies. The best way to do this is by using your... background, the more colonies you will need to screen.   Saving clones for later culture: At this point...

  14. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...ve learned that increased accessibility is achieved through interconnected incremental changes. It was...When Addgene opened in 2004, we focused on creating a repository that made it easy to share plasmids,...identified and offered a number of solutions that increased accessibility, such as using couriers that refill...In some cases, the distributors broadly helped increase access in the country, and we can see a growth...re continuing to use our data and resources to increase accessibility to researchers around the globe....the materials in our repository. We started to increase accessibility to our repository by making it easier...Bacterial Transformation How to Design a Primer Creating Bacterial Glycerol Stocks Inoculating a Liquid...

  15. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

    Type
    Blog Post
    ...article.) Modifying AraC to create BLADE The team modified AraC to create BLADE by replacing its dimerization...images created by the team (Fig. 2). These images are based on the concept of bacteriographs created about...bacteriograph of Michelangelo's "The Creation of Adam." This bacteriograph was created using 160 individual images...dimerize and activates transcription. The team created two constructs, among others: pBLADE-mCherry:...about a decade ago. To create these images, the scientists used a strain transformed with pBLADE expressing...instead of mCherry under the PBAD promoter. They created a bacterial lawn on an agar plate, put a mask over... that Di Ventura’s lab and Khammash's lab have created and shared with Addgene. You can find all the plasmids...

  16. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ...Cancer cells find a way to grow haywire, access and create more vasculature to feed themselves, use the blood...the tumor microenvironment - a vital atmosphere created by the cancer cells to shield themselves from attack...cells of epithelial origin. Suppressing function, creating tolerance, and evading recognition are a few methods...cells into regulatory cells to suppress function. Creating Tolerance T-regs in the tumor microenvironment...HIF-1), which promotes the production of VEGF to increase angiogenesis around the tumor. This in turn regulates...signals released from the microenvironment help create tolerance beyond direct proximity to the tumor;...cells (DCs) and macrophages (M1 and M2) that help create welcoming environments for tumor cells spreading...

  17. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...mutations increased the accuracy of editing by up to 10-fold per allele. This is a 100-fold increase in editing...incorporate homo- or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking mutations...find the 0.2-0.5% correctly edited alleles, when screening reasonable amounts of single cell clones. Furthermore...tedious business of iPSC clone picking, a 100-fold decrease is the difference between picking 100 versus 10,000... together with the intended sequence change and screen a few hundred clones to find the right one Step... few hundred. From these two steps, you’ll be screening a total of a few hundred clones - Still better...efficiency of mutation incorporation falls with increasing distance from the cut site. If the distance is...

  18. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...recombination, increases plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability...that make major improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below...sensitive. dnaJ Mutation in a chaperonin gene Increases the stability of certain expressed proteins ...methylate DNA lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced...derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1 galE15 galK16...cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR...General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient; good strain for...

  19. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ...encoding Gag and Pol, increasing the number of recombination events necessary to create a replication-competent...Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...shows how the lentiviral genome was condensed to create the 2nd-generation lentiviral system (Figure 2)...safer than the original system, the possibility of creating a replication-competent virus via recombination...possibility and enhance biosafety, Dull et al. created the 3rd generation system (Figure 3), which differs...Lentiviral vectors in the lab Lentiviral vectors are incredibly popular in the lab - the Trono lab’s lentiviral...both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since lentiviral...

  20. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...CBEs). But unlike CBEs, CGBEs then excise the U to create an abasic (empty) DNA site using either fused or...nucleobase component of the nucleoside inosine. Created with BioRender.com. Inosine excision leads ... able to edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this... for A → X base editor (X = any nucleotide). To create a more specific A → C base editor (ACBE), they ...Black arrows indicate natural cellular processes. Created with BioRender.com. The first deaminase-free...either to directly accomplish the base edit or to create the substrate for a base excision repair pathway...nCas9. First, MPG removes the G base from DNA to create an abasic site. Like in most other base editor ...
Showing: 281 - 300 of 957 results