We narrowed to 888 results for: URE

...engineering enzymes, each with their own unique features and quirks. Here, we will review the genome engineering.... Dozens of variants exist with unique cutting features and evolved PAM sites, making Cas9 an adaptable...PAM does not require any G’s or C’s. One curious feature of this Cas is that once it is engaged by its target...target, it will non-specifically cut ssDNA. This feature is shared by other Cas enzymes outlined below as... proteins should not be overlooked. The unique features and targeting abilities of the other Cas family...Janice, et. al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362(6416): 839-...with the single-protein CRISPR effector Cas7-11. Nature, 597, 720-725 (2021). 10.1038/s41586-021-03886-...

...the analysis did find 263,000 more osteoporotic fractures. The overall economic savings to the US healthcare... Unfortunately, it also meant that, by the very nature of the way the experiments were performed, they...and cell research. Using cells from both sexes ensures that the diagnostics and therapeutic interventions...recent years, and you can see a summary of this literature in our health and medicine checklist. A common...not analyzed) may harm humans or contribute to failures in drug development – costs far greater than those...doing the research correctly from the get go. The future of sex and gender in research There is much work...Blog Learn How Scientists Are Trying to Shape the Future of Research Learn How Notions of Scientific Impact...

...individual parts are reusable, saving time and money on future assemblies. If you are new to MoClo, give our MoClo...synthesis of these molecules. To make large amounts of pure product in an affordable way, researchers test many...your yeast experimental goals are more general in nature, or you aren’t planning on using K. phaffii, then...proteins within the mitochondrial matrix.     Figure 1: A chart with MoClo kit recommendations for different...2023). The Plant Parts II kit has a focus on infrastructure with shuttle vectors, several delivery systems...targets or multiple sgRNAs against the same target to ensure efficient cutting. Beyond sgRNAs, MoClo can also...convolute the results of these experiments. The modular nature of the Fragmid system allows for direct comparisons...

...Behavioral experimentation I’ll discuss some of the key features of each of these steps. Today we’ll discuss selection...relies on identification of the bregma and lambda sutures and subsequent determination of the proper coordinates... your brain region of interest (as shown in the figure below). Since most of the precision of optogenetics...circuits underlying brain disease in animal models. Nature reviews Neuroscience. 2012;13(4):251-66. doi: 10.1038...of neuroscience. 2011;34:389-412. doi: 10.1146/annurev-neuro-061010-113817. PMID: 21692661.  3. Bernstein...direct comparative analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808...mice for light-induced activation and silencing. Nature neuroscience. 2012;15(5):793-802. doi: 10.1038/...

...we’re really borrowing these tools from Mother Nature’s toolbox. Natural plasmids, also called environmental...the basis of many molecular biology tools! Figure 1: Common functions of natural plasmids. Created... to survive where others can’t. This ability to endure a harsh environment is distinct from the benefit...new types of plasmids without clear analogues in nature. For example, helper plasmids were developed to... To learn more about some other common plasmid features, such as origins of replication and promoters,... blog as scientists continue to explore Mother Nature’s toolbox for new and exciting molecular technologies...Resources References Demanèche, S., Bertolla, F., Buret, F., Nalin, R., Sailland, A., Auriol, P., Vogel,...

...Did you say… easier? Fragmid’s modular assembly structure takes a lot of the design work out of the plasmid...Fragmid components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the...schematized. Sizes of fragments are not to scale. Figure and caption from McGee et al., 2024, used under...designing a CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...website.   In step one, you’ll select the features of your vector, which are: Organism (Mammalian...mechanism (KO, CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website...want? You can make your own Fragmid components! Ensure your backbone and genes contain only the Bbsl cut...

...focus was on editing animal models of disease and cultured cells. For example, in 2013, Jinsong Li and colleagues...When all was said and done, the researchers had cured 24 mice of their disease. Meanwhile, Hans Clevers...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for...success rate in clearing misfolded protein levels as measured in the blood. More information is expected soon...blindness, and diabetes. Directions and obstacles for future CRISPR therapies CRISPR technology is rapidly evolving...strides since we first wrote this blog post. The future success of CRISPR in the clinical will rely on ...MA, Berkhout B, Herrera-Carrillo E. A CRISPR-Cas Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(2):1563...

...office or other authorized office directly to make sure the deposit agreement/MTA is taken care of and, ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit...Required columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have...Growth Temp Choose from 30 °C, 37 °C, or room temperature. Growth Strain Please indicate which growth...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the...update the spreadsheet every so often and this ensures you are using our most up-to-date version! We hope...

...(ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ~ 100nm...genomes/mL) can be measured by: Optical density: particle concentration is measured by looking at absorbance... construct from step #1 and allowed to stay in culture for up to 20 additional days at which time the ...recombinant adenoviruses using the AdEasy system." Nature protocols 2.5 (2007): 1236. PubMed PMID: 17546019...production of high-capacity adenoviral vectors." Nature protocols 4.4 (2009): 547. PubMed PMID: 19373227...

...Met protein complementation. In the procedure diagrammed in Figure 3, students first transform the S. ...available (6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway ... the deletion). Find the Plasmids Here Plasmid features enable multiple student experiments We used TOPO...coding sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/... controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to the C-termini...expression by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...

...sequencing (NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created... capsids, or serotypes, have been identified in nature, while many more have been engineered in labs. ...improve infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes...smaller Cas9 ortholog like SaCas9. You could even venture away from the comfort of Cas9, and opt for a completely...design and execution of CRISPR in vivo screens. Nature Protocols, 17(9), 1903–1925. https://doi.org/10.1038...vector integration into CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/... linkage analysis with in vivo AAV-Perturb-seq. Nature, 622(7982), 367–375. https://doi.org/10.1038/s41586...

...electrical potential. Thankfully, there is a tool to measure these electrical events! Voltage indicators are...They can be targeted to the plasma membrane to measure membrane potential and can also be directed to ...applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet is a fusion between the voltage-sensitive...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically...vitro and a small molecule is added exogenously to measure a phenotypic response in the form of fluorescence...Indicators As previously mentioned, cellular voltage is measured at the cell membrane, and this voltage is particularly...has you covered. Happy voltage sensing!   Figure 3: Components of the types of voltage indicators...

...compatibility and high DNA specificity." Nature (2018) doi: 10.1038/nature26155 PubMed PMID: 29512652 Plasmids...loci or genomes. Cas9s from other species, like S. aureus and N. meningitidis require different PAM sites...first restored catalytic residues D10 and H840. To ensure that the clones were capable of cleavage, they ...fused to third generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing efficiency...efficiencies (edit frequency in cell population measured with NGS) are shown as percentages, with fold ...lot we still don’t know about xCas9. “I’m not 100% sure xCas9 is going to be flat out better than SpCas9...

...the CRISPR/Cas9 system. Nature 516:423–427 . https://doi.org/10.1038/nature13902 Maggio I, Holkers M, Liu... the target cells (Beatty et al., 2012). These features make AdV an attractive candidate for in vivo delivery...genome editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) ...Gonçalves' lab found in a second paper published in Nature Methods the same year that delivering RNA-guided...double-stranded AdV genomes. Such protein-DNA structures presumably reduce the likelihood that donor DNA... that advantages of AdVs include their episomal nature and very efficient introduction of DNA into therapeutically...

... to quickly check out the plasmid's structural features and see if I can easily clone my gene into it?...plasmid map based off of the plasmid sequence and feature information given by our depositors. Many depositors...plasmids are sent as bacterial stabs at room temperature. Core of the plasmid page - Core of the plasmid...), antibiotic resistance, and optimal growth temperature are listed, as well as the name of the strain... Citing this Plasmid: This is a relatively new feature of our plasmid pages, and provides a list of publications...our blog post) for more information about this feature!).  This means you can see which labs have already...the Addgene Blog Read about Our New Citations Feature Check out Our Walkthrough of the Deposit Process...

...exclusively to in vitro cell culture. The user should review the current literature about their preferred model...single-stranded DNA oligonucleotide, so you will need to make sure your expression system and delivery methods are ...system, a good first step would be to consult the literature to see if anyone has published a protocol that...in in vitro transcription reactions to generate mature Cas9 mRNA and gRNA. RNA is delivered to target ...different methods, so we've summarized some key features of common  delivery methods below: Chemical transfection...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn about CRISPR Delivery Using Ribonucleoproteins...

.... Figure 1: Like this jar of jellybeans, a pooled CRISPR library is a complex mixture. Pooled libraries...experimental system are absolutely necessary to ensure that you select the correct CRISPR library. Choosing...concentration that is sufficient to generate lentivirus. Be sure to use next-generation sequencing to check the quality...cells gain resistance to drugs and can identify future therapeutic targets for diseases causing uncontrolled...uncontrolled cell growth, such as cancer. Figure 2: The CRISPR library must be amplified (A) to be ...the screen should be independently validated to ensure that the desired modification produces the phenotype...

...causing a lack of exposure, a lack of immunity, and then finally a synchronized exposure under these conditions...conditions once the measures were lifted (Servellita et al., 2023).  This anecdote goes toward saying that...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from...31749409. Harrach, B. (2014). Adenoviruses: General Features. In Reference Module in Biomedical Sciences. Elsevier... Undergoing Spontaneous Degeneration in Tissue Culture. Proc Soc Exp Biol Med. 84(3), 570–573. https:/...type 2 in US children with acute severe hepatitis. Nature, 617(7961), 574–580. https://doi.org/10.1038/s41586...

...re tethered to bring them into close proximity (Figure 1). Formation of the complete FP results in a fluorescent...can be detected by fluorescence microscopy. Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent...localization of the studied PIPs. The straightforward nature of BiFC also makes it easier to generate a large...fragments and, preferably, some information about the structures of the tested PIPs. Many split FPs have been ...BiFC experiments only require that you image and measure the fluorescence of a single FP. Your control construct...one another — which means it cannot be used to measure the dissociation of PIPs. Thus, whereas BiFC is...fluorescence complementation (BiFC): A 5-year update and future perspectives. Biotech 53. https://doi.org/10.2144...

...center of the structure, shielded from quenching by aqueous solvent. This tightly-packed structure explains...Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish for millions of years; however...while still maintaining fluorescence. The protein structure, first reported in 1996, is an eleven β-sheet-...yield for BFP Y145A and H148D Stabilizes the structure of Cerulean derivatives F99S, M153T, V163A Improves...Optogenetics: Scientists can use light to detect, measure, and control molecular signals, cells, and groups...This is a type of flow cytometry that separates mixtures of cells into distinct populations based on fluorescent...