...Control of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Sajwan S, Mannervik...and Fluorescence Imaging. Cell 159:635–646 . https://doi.org/10.1016/j.cell.2014.09.039 Zhou H, Liu J,...from when looking to activate genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation...large and are not expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex ...activation levels than SAM in some loci across Drosophila cell lines (Sajwan et al., 2019). SPH SPH combines components...

...Inhibitor of CRISPR-Cas9. Cell 170:1224-1233.e15 . https://doi.org/10.1016/j.cell.2017.07.037 Li C, Psatha...Off-Switches for CRISPR-Cas9. Cell 167:1829-1838.e9 . https://doi.org/10.1016/j.cell.2016.11.017 Shin J, Jiang...concern is that the longer Cas remains active in a cell, the greater chances there are for off-target edits...within the first six hours. When added to human cells six hours after the introduction of Cas9 RNPs, AcrIIA4...CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells. Molecular Therapy - Methods & Clinical Development...

...genes should be ‘read’ by the cellular machinery. This is how most cells in the body contain the same ...complex of DNA and histone proteins that compacts cellular DNA into chromosomes and reduces the risk of dangerous...the more compacted it becomes. This prevents the cellular machinery from physically interacting with the...visualise changes in histone methylation in living cells. Essentially this reporter construct consists of...recognise lysine 27 methylation. When tested in living cells where methylation of these sites was controlled,...fluorescent reporters of histone methylation in living cells." Journal of the American Chemical Society 126.19...

...unwinding steps. Molecular Cell, 70(3), 385-394.e3. https://doi.org/10.1016/j.molcel.2018.03.031 Xiao, Y., ...CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5. https://doi.org/10.1016/j.molcel.2023.01.024 Morisaka, ...there isn’t a template strand left behind that the cell can use to repair. Figure 3: General overview...broad and unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038...CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12), 1471–1477. https:...

... for EM is not something you find in the average cell and molecular biology lab, and EM is limited in ...ultrastructural relationships that influence everything from cell division to memory, but don’t have access to the...fix (and maybe label) your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample...properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain ..., Mohar B, Eddison M, et al (2022) Visualizing cellular and tissue ultrastructure using Ten-fold Robust... Zwettler FU, Guennec ML, et al (2019) Imaging cellular ultrastructures using expansion microscopy (U-...2016) Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins...

...base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j.molcel.2024.01.021 Tong, H.,... edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. The...Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template... efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650...efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https:...

...Generating a no-fuss genetic knockout  Knock-out cells or animals are engineered by introducing Cas9-mediated...transcription by blocking its initiation. In mammalian cells, robust repression requires targeting dCas9 along...study essential genes whose knockout would cause cell lethality. The Krüppel associated box (KRAB) is ...repressing target genes in a variety of species and cell types can be found on Addgene’s website. Activating...activating target genes in a variety of species and cell types can be found on Addgene’s website. ...optimized for expression in different species and cell types including (but not limited to) bacteria, yeast... have delivered Cas9 and a sgRNA to your target cells, it is time to confirm that your target sequence...

... infected cells to go undetected by the immune system in a novel way, by shielding host cell surface proteins... itself to escape vesicles within host cells and turn the cell into a veritable virus factory.  Their ... sets off.  The virus works its way into immune cells in ways that aren’t fully understood. Much of the... viral attachment and fusion. Once inside a host cell, new evidence in PLoS Pathogens shows that the Ebola...

...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...transformation of chemically and electrocompetent cells is minimal (picogram range). It can be extracted...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...process of inserting an external plasmid in mammalian cells and it will likely require micrograms of plasmid...

...Neuroscience at Harvard University. Just as computers, cell phones, and cars become more technologically advanced...gene mutation is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with... of the mutated β globin gene that causes sickle cell anemia. The mutation happens to destroy a sequence...conducted on a sample from an individual without sickle cell anemia, two DNA fragments are observed because the... site where MstII cuts. In a patient with sickle cell anemia the enzyme does not cut the DNA so only one...Wai Kan. "A sensitive new prenatal test for sickle-cell anemia." New England Journal of Medicine 307.1 (...transgenic constructs and mice." Current Protocols in Cell Biology (2009): 19-10. PubMed PMID: 19283728. PubMed...

...interest live within a cell,” “Does this disease change the architecture of my cells,” or “How does this ...IF), is an immunoassay that brings to light the cellular world. The technique allows you to ask questions...this mutation impact the types of cells found in my tissue.” It is based on the same principles as other ... visualize a target of interest within an intact cell, tissue, or, in some cases, a whole organism. But...joined to fluorescent molecules are applied to a cell or tissue sample where they bind to their targets...to visualize a target within an intact sample, a cell in the case of ICC or a tissue in the case of IHC...

...partners in mouse cell lines. Dr. Di Schiavi hopes that these studies will accelerate the development of...learn how plasmids from Addgene will be used to accelerate rare disease research and provide hope to patients...plasmids to upregulate their expression in specific cells. With a more concrete understanding of the molecular... of ~30 patient derived peritoneal mesothelioma cells lines and assess their tumorigenesis. These experiments...effects on the disease. This work will hopefully accelerate the development of targeted therapy for peritoneal...

...millions of cells. For every individual sgRNA, you recover a minimum of 1,000 targeted cells to sequence...trying to generate a knockout cell line, with the goal of isolating single cell clones to use for multiple...generate a ssDNA break which can easily be repaired by cells with a near zero likelihood of mutation arising.... a level of, say 5%, then the majority of single cell editing events will be on target. Thus, if you isolate...example, if your goal is to generate a knock-out cell line to perform downstream characterization of, ...

...at lower copy numbers within bacterial cells, and bacterial cells with multimers grow more slowly compared...stable and more prone to being lost in bacterial cells compared to monomers, although this can be prevented...increase the frequency of base substitution in human cells. J Biol Chem. 2011 Mar 25;286(12):10017-26. doi:...plasmids leading to the formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674...supercoiled plasmid DNA concatemers in mammalian cells. J Gene Med. 2009 May;11(5):444-53. doi: 10.1002...essential for plasmid monomerization and stability. Cell. 1984 Apr;36(4):1097-103. doi: 10.1016/0092-8674...

...path.   Yeast as a model organism Yeast are single cell, eukaryotic organisms formally classified as fungi...and S. cerevisiae (budding). Yeast make for an excellent model organism for a few reasons: Affordable...similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a number ...protein bang for your media buck - it grows at higher cell densities than most of its counterparts, yielding...oxidative reactions. They are found in all eukaryotic cells and peroxisomes are where methanol metabolism occurs...in methylotrophic organisms. K. phaffii is an excellent model for peroxisome studies since their peroxisomes...

... can rupture E. coli spheroplasts (cells which have had their cell walls removed with lysozyme). This ...protein fusions allowed us to image R bodies inside cells, and these constructs may be useful to future scientists...applications. For example, you could use them to lyse cells (and surrounding membranes) in keeping with their...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond...assemble and function, we might be able to endow cells with new ways to interact mechanically with their...

... used HEK293T cells in our first study, but we're planning to soon expand to other cell types, including...26 of the 29 SARS-CoV-2 viral proteins in human cells and used these viral “bait” proteins to fish for...human proteins that the virus interacts with in cells. Because viruses rely on the help of human proteins...monkeywrench into the virus’ hijacking of human cells. Even better, more than half the drugs hitting these...they have an IRES-Puro marker to facilitate stable cell clone selection. Expression is driven by a fairly...

...different types of human cells, including T cells and hematopoietic stem and progenitor cells. Now with the DAP...to enable multiplex gene editing in a variety of cells and organisms. However, lengthy genetic payloads... over 50% averaging editing efficiency in human cells. MBE with DAP array also exhibited reduced Cas9-... editing, and nuclease editing, in virtually any cell type or organisms of interest with only minimal ...

...genetic cargo, typically an expression construct, to cells/organisms. This is often cDNA, shRNA, or something...perform very similar functions to infiltrate the host cell and deposit viral cargo. Gene expression While AAV...what will be delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry...altered tropism of AAV to target different tissue or cell types. AAV also requires some back up from adenovirus...promoter that you wish to deliver to your target cells. In adenovirus, the ITRs serve as a type of origin...deficient. This is typically supplied by the production cell line (HEK-293) during production. Fun fact: There...

...to quantify rates of mutation in a bulk or single cell-derived genomic DNA sample as part of a precision... successfully analyzed gDNA from a few thousand cells from single wells of a 96-well plate without prior... that they are easily able to distinguish single-cell derived samples with mono-allelic mutations versus...Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Meth 11, 291–293... and Highly Specific Screening of Genome Edited Cells. PLoS ONE 11, e0153901–17 (2016). PubMed PMID: 27089539...mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31, 822–826 (2013). PubMed...