We narrowed to 805 results for: Cre

...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...

...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...

...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...

...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...

...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...

...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....

...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 ...

... 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...

...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...

...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...

...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...

...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...

... KJ’s successful base editing intervention was created. Only about half of babies with KJ’s disorder survive...survive long enough to receive a liver transplant — creating intense time pressure for the research team. Ultimately...base editing advances that led to KJ’s treatment. Created with BioRender.com. Invention of base editing...DNA-targeting technology with a fused cytidine deaminase to create C→T base edits at a target locus. A year later,... (Gaudelli et al., 2017). These editors did not create double-stranded breaks in the DNA backbone, making... be targeted (Walton et al., 2020). The initial screen for base editors that could target KJ’s mutation...PAMs available, a large number of gRNAs could be screened for the most efficient editing. In these initial...

...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...

...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...

...Marker B. Created with biorender.com.   The goal of flow cytometry is to use increasingly more stringent...populations that are B+ and one population that is B-. Created with biorender.com.   If it’s hard to conceptualize...imagining (or drawing) a quadrant over the dot plot. Created with biorender.com.   Of course, this is quite...al., 2017 to identify mDCs and pDCs. Used under Creative Commons license.    In the third plot, anti-CD14...visualize the different populations. Used under Creative Commons license.    Pro tip! Subtypes of immune...the percentage of cells in each gate. Used under Creative Commons license.    Contour plots  FACS data...data from Jhunjhunwala et al., 2015. Used under Creative Commons license.  Histograms  For analysis...

...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 ...

...in therapeutics, as it can increase gene expression at certain loci to create changes in phenotypes. For... storm. While Cas9 has been most widely used to create specific edits in DNA, there has also been significant...inducible upon environmental triggers. This is done by creating a split dCas9 protein that can reassemble when...the best uses for Cas9 activators is in genetic screening. gRNAs targeting every gene in the human genome...interactions. Additionally, large scale CRISPR-based screens can be used to gain insight into protein structure...focused advancing scientific discovery through the creation of innovative technologies. To date his group ...of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568:511–516 . https://doi.org/10.1038/s41586...

...nuclease domains, researchers created the CRISPR nickase. Nickases create a single-strand rather than a...generated by the nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design...Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving nickase. Instead of...strands bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs...other? The gRNAs must target different strands to create a DSB, but this can be accomplished with either...an individual gRNA with WT Cas9, repair levels decrease rapidly 10 bp from the cut site. So if you can... you can’t obtain high HDR efficiency. Nickases create a staggered cut - could this system mediate repair...

...autophagosomes with lysosomes leads to an increase in yellow puncta but a decrease in red puncta. Find the mRFP-...RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence suggests an increase in autophagic...flux, while an increase in GFP fluorescence relative to RFP fluorescence suggests a decrease in autophagic...yeast or the lysosome in mammalian cells. This decrease in pH quenches SEP but not DsRed, leading to emission...Therefore, the formation of autophagosomes leads to an increase of yellow puncta (GFF positive/RFP positive), ...fusion of an autophagosome with a lysosome. An increase in autophagy leads to more yellow and red puncta...