...available empty backbones, visit Vector Database. Step 2: Insert name  Include information about the insert...selection markers (for use in other cell types, e.g. eukaryotic cells). pBACKBONE-XXXXX Tip: For a catalog...

...faster when using a dual guide system.   Fig. 2: Size screening for large deletion events.   ... Biol 17: https://doi.org/10.1186/s13059-016-1012-2 Find more CRISPR Resources at Addgene: Read our CRISPRGuide...number of cells have been edited. For many experiments, the end goal is to generate a clonal cell line, so...designed your gRNA and introduced it into your target cells with Cas9. Hooray! Now it’s time to make sure your...blog post we’ll explain how to verify that your cells were appropriately edited for your desired mutation...important to know a few things about your target cells and the desired edits. First, you will need to know.... Validating knockout mutations When a knockout cell line is desired, any mutation that introduces a ...

...the chromosome, ending replication.   Figure 2: Prokaryotic chromosomal replication. Adapted from...Microbiology and Molecular Biology Reviews: MMBR, 62(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...

... 24336571. PubMed Central PMCID: PMC4089965. 2. Sanjana, Neville E., Ophir Shalem, and Feng Zhang. "Improved...both Cas9 and your gRNA of interest to mammalian cells. It was designed to have increased lentiviral titer...knock-out thousands of genes in mixed population of cells as part of pooled library screens (1,2). The human...pooled libraries targeting both mouse and human cells are avaliable for distribution. To learn more about... includes work probing mTOR signaling in cancer cells, deciphering the mechanism of matrix metalloproteinase...of the many plasmids being distributed to and accelerating research in labs all around the world. We thank...Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87. PubMed PMID: 24336571...

...sequence is unique compared to the rest of the genome.  2) The target is present immediately upstream of a Protospacer...utilized allowing for PAM flexibility. Fig. 2: Prime editor engaging target DNA. The reverse transcriptase...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. ...

...extraction solution, runs the protocol, then uses around 2-4ul in each 50ul PCR reaction. The gDNA extracted ...find both single-allelic and bi-allelic cells. Single-allelic cells usually make up the majority in culture...specificity. A good reference is this Cell paper: http://www.cell.com/abstract/S0092-8674(13)01015-5 As for.... A good example/reference is this Cell paper: http://www.cell.com/abstract/S0092-8674(13)01016-7 In the...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....CRISPR is used to engineer a locus, isolate single cell colonies, then genotype them, it is possible to ...or delivery the two plasmids together into your cells. If you want to use double nickase, you need to ...

...Intro Chapter 1 - Who are Addgene's Founders? Chapter 2 - The process of founding Addgene, where the idea ...co-founder of Addgene. Melina received her Ph.D. in Cell Biology from Harvard University, where she studied...Plasmids Find Plasmids for FRET Find Plasmids for Subcellular Localization ...

...tagging." Nature methods (2015). PubMed PMID:26322837. 2. Stadler, Charlotte, et al. "Immunofluorescence and...that inntags IT5, IT6, and IT10 expressed well in cells and did not form undesirable aggregates. Using budding...tags. Impressively, IT6-GFP did not reduce yeast cell size or viability compared to untagged GFP overexpression...multiple other proteins. Again, little to no change in cell morphology was observed with IT5 and IT6 fusions...and HA tags had especially detrimental effects on cell size and growth rate, indicating that they compromised...correlation for protein localization in mammalian cells." Nature methods 10.4 (2013): 315-323. Pubmed PMID...

... both substantial merit and national importance; (2) The foreign national must be well positioned to advance...outstanding achievements in your specific academic field, (2) that you have three 3 years of research experience..., Casseus Law suggests a mix of experts including 2 dependents –people who have worked with you i.e. your...This post was contributed by guest bloggers Lauren Celano of Propel Careers and Rachel Casseus, Esq. Founder...below. An international or national award for excellence in your field. Membership in a professional organization...counsel. Many thanks to our guest bloggers Lauren Celano of Propel Careers and Rachel Casseus, Esq. Founder...resident green cards and citizenship.   Lauren Celano, CEO Propel Careers. Lauren is passionate about...

...Halloween is only 2 days away and here at Addgene we couldn’t be more excited! As a small, tight-knit...teamwork, and competition. And our Halloween celebration this Friday will combine all of these. Addgenies...

...mammalian cells, or in purified form, and didn’t need any added cofactors.      Figure 2: Fluorescence...of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled...

...double strand break), increasing editing efficiencies 2-3-fold. However, the process of double nicking increases... editing as efficient as possible. Figure 2: Decision tree for selecting a PE6 variant. PE = prime...using paired prime editing. Nature Biotechnology, 40(2), 218–226. https://doi.org/10.1038/s41587-021-01025...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...efficient prime editors. Cell, 186(18), 3983-4002.e26. https://doi.org/10.1016/j.cell.2023.07.039 Lin, Q.,...correct only a minority of those variants in most cell types. But prime editing, a CRISPR technique developed...unedited DNA. The heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing...

...of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC...deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that activates...the mesocorticolimbic system in addiction. Front Cell Neurosci. 2014;8:466. PubMed PMID: 25653591. PubMed...

...’re delivering their payload to.   Figure 2: Numbers of AAV tools, by category, available from... substrates, light, or engineered ligands (Figure 2). Biosensors, which include calcium and voltage sensors...As part of Addgene’s 20th anniversary celebration, we’ve been finding and sharing stories unique to our... be safely used to deliver a genetic payload to cells, and the complexity of effectively deploying them... can be to use. AAVs, which can target different cell types depending on their serotype, are particularly...

...Information Science and Technology 60.4 (2009): 858-862. 2. Van Noorden, Richard. "Interdisciplinary research...Librarianship and Scholarly Communication 2.2 (2014): 2. Additional Resources on the Addgene Blog 6 Tools... the 926 video abstracts identified. The journal Cell had the largest average view count per video abstract.../wesharescience.com/pin/7472 Article: http://www.cell.com/current-biology/abstract/S0960-9822(16)30873...

...First, the light-responsive molecules of cryptochrome 2 are the chromophores flavin-adenine dinucleotide and... vectors and genetically target them to specific cell populations. They demonstrated use of the LITEs ...non-invasively, its use is not restricted to cultured cells but extends to freely moving animals, as established...novel mode of optogenetic control of endogenous cellular processes." The LITE system can now be applied...no means limited to particular model organisms or cell types. “This tool is really flexible and modular...

...which would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore...population.     Figure 2: Dump gating labels all unwanted cell lineages with the same fluorophore...out all CD8+ T cells from a mix of CD8+ T cells, CD4+ T cells, B cells, and dendritic cells. In this case...+ T cell, dendritic cell, CD8+ T cell. Created using biorender.com.   Intracellular labeling Oftentimes...in flow cytometry are cell proliferation and cell viability. Unlike intracellular labeling, dye staining...be done on live cells. For cellular proliferation, esterified dyes that can enter cells freely are typically...amines on the cell surface, which yields a much weaker signal on live cells compared to dead cells. As a result...

...chosen SspB-tag-GEF and your cells will be ready for the spotlight.    Figure 2: Opto-RhoGEFs to control ...to a specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID ... target to specific subcellular regions, laser ablation can damage other cellular structures, and genetic...demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given the long ... expressed in a monolayer of endothelial cells, showing cell protrusions and overlaps (arrows) after blue...2016). Rho GTPase signalling in cell migration. Current Opinion in Cell Biology, 36, 103–112. https://doi.org...Optogenetics gives you the power to control cells or organisms with the flip of a switch. You may be ...

...microdomains of live cells." Science 296.5569 (2002): 913-916. PubMed PMID: 11988576. 2. Tsien, Roger Y. ...phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production can be detected by using a MS2 coat...proteins to study mRNA trafficking in living cells." Methods in cell biology 85 (2008): 273-292. PubMed PMID...genomic loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed... fluorescent protein in almost all organisms and cell types, (ii) no need to add a co-factor, (iii) easy... the bathing solution and require uptake by the cells. Which of these strategies works best for a specific...showing that its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles...

...using a 2-step PCR-based protocol and are compatible with single-end or paired-end read flow cells. FREQ-Seq...wheat germ, HeLa, and Leishmania (Gagoski, et al 2015[2]). The proteins encoded in this set range in size ...Purification Process with pCri Cell-free protein expression kit Cell-free expression systems have been...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of...testing and comparison of different cell-free expression systems; the Cell-free expression test kit represents...: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein Expression Kit FREQ Seq PUF Assembly...facilitate gene expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The expression...

...Calcium sensor Ting 83899 pAAV-mDlx-GCaMP6f-Fishell-2 mDlx GCaMP6f expression under the control of the mDlx... of Cre, and expresses NLS-EGFP in Cre-positive cells. Control Harvey 137164 pAAV-nEF-Con/Fon/Von eYFP...

... a template, copying the base edit. Figure 2: Cytidine deamination takes place on the free strand...SpCas9 base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated...formation. To further decrease indel formation 1.5-2 fold, the team fused bacteriophage protein Gam to ...that it appears “newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand..., which may lead to cell death rather than NHEJ repair, thus removing these cells from the edited population...target modification in primary T cells, making them a promising tool for cell therapy applications. Dual base...relatively low frequency, especially in nondividing cells. Scientists everywhere wistfully imagined being ...

...caeomi>2.0.co;2 Uphoff CC, Drexler HG (2011) Detecting Mycoplasma Contamination in Cell Cultures by Polymerase...bringing in environmental contaminants.  Figure 2: An Addgenie works in the tissue culture hood while...mycoplasma contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas...present in most of the cell culture facilities, tissue culture labs and every cell culturist has to deal...cost-effective (Sung, 2006). The sensor cell-based method uses specialized cells that can detect the presence of... should be made available for cell culturing and maintenance of the cell lines. Providing such an environment...Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma contamination...

...another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...when (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets...look at the subcellular localization of a protein of interest. Conveniently, you found a cell line in which...antibody binding in the transfected cells but not in control cells. But (there is always a ‘but’), you...transfect it into your favorite cell line. In parallel, you transfect cells with another plasmid not encoding...the cells transfected with your protein of interest and show just the faintest band from the cells transfected...when you start using complex samples such as whole cell lysate or tissue sections. It is important to confirm...

... a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using...expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of...readout for subcellular fractionations, a procedure that isolates proteins from different cellular compartments...interest from Addgene, transfected it into your target cells, now what? How can you tell if the protein you are... are so keen to study is expressing in your cells? Immunoblotting or simply the western blot, or western...specific protein within the complex mix derived from cells or tissues. Westerns are useful not only to detect...protein levels relative to standards, detect the cellular location of proteins, and can be a readout for...

...posts: Part 1: Genetically Engineered Mice and Part 2: Breeding and Crossing Mice. My first CRISPR mouse...is less predictable. In our best case, we saw that 2 out of 3 mice carried a 5 kb insertion in the ROSA... into a mouse embryonic stem cell (ESC) line, enrich and select for cells that had successfully incorporated... when it gets exciting – the cell sends out an “SOS” signal and cellular repair mechanisms rush in to ...Cas-Mediated Genome Engineering. Cell 154:1370–1379 . https://doi.org/10.1016/j.cell.2013.08.022 Yoon Y, Wang ...Cas-Mediated Genome Engineering. Cell 153:910–918 . https://doi.org/10.1016/j.cell.2013.04.025 Zhang X, Liang...develop into chimeric mice (with a fraction of the cells in the adult mice derived from the engineered ESCs...

...Drosophila melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169...attaches to the cell wall of green algae (Soo et al., 2015). There, it divides into two cells. At the same...Vampirovibrio). What’s left of the algae cell is just the cell wall and cytoplasmic membrane. Vampirovibrio...

... or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA. Edited cells may be...expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard... CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may...identify and enrich positive cells, or selection marker to generate stable cell lines (for details, see Plasmids...wide variety of mammalian cell lines Useful for difficult to transfect cell types, can also be used in...possible genotypes within the resulting cell population. Some cells may be wild type due to either (1) a ...will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR ...

...similar optical quality (magnification of 140X and 2 micron resolution). I have worked many hours in a ... to microbes by using non-invasive methods for cellular imaging with ultrasound. In his research he applies...

...access to the results of federally-funded research. 2)      In June, the White House honored 13 “Champions...Change,” including our recent guest Drew Endy, in celebration of open science. 3)      Also in June, the G8...

...you are having trouble preparing your most excellent resume. 2) Follow job boards and check them regularly...give you a chance at an interview:  1) Have an excellent resume ready to be submitted The structure and...successful application. It takes time to prepare an excellent resume…it is worth it. Read here to get help if...

...Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1. Image from Taghbalout...for studying the bacterial cell cycle, asymmetric cell division, and cellular differentiation, but it takes...crescentus Multiplexed gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors...complex remains in the cytoplasm. Read the paper in Cellular and Molecular Bioengineering Find the optogenetic...Figure 1: Serial dilutions of CRISPRi in Caulobacter cells for ctrA gene expression using various dCas9. Image...lab repressed ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and...piggyBac transposon system for human pluripotent stem cells Lindy Barrett’s lab developed new fluorescently ...

...photoconvertible fluorescent protein. Nat Methods. 2009 Feb 6(2):131-3.  PubMed. Find plasmids from this publication... are you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct...shown to work in plant, Drosophila and mammalian cells, and it may find wide use in biological research...turnover rate. In many cases, however, a GFP-tagged cell line or organism has already been made, and it would...made by accident. After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. ...spontaneous conversion did not occur. Tobacco suspension cell culture with cytosol-targeted GFP. White circles...this technique may not be appropriate for high-pH cellular compartments. Sattarzadeh et al. have shown that...

...Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of the mouse brain. Rubin...recombination varies for different constructs and cell types. Second, Cre may induce recombination at pseudo...standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding domain-fusion...recombination efficiency. When expressed in neuronal cells, each line showed broad recombinase-dependent RFP...these recombinases were also active in mammalian cells. In experiments, B3 and KD were shown to be functional... damage induced by Cre recombinase in mammalian cells. Loonstra A, Voojis M, Beverloo HB, Al Allak B, ...recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation. Heidmann...

...might otherwise be hard to identify. Figure 2: Users can cross-interrogate screens from the literature... disease, cell type or drug treatment can browse through screens performed in relevant cell lines or conditions... currently houses over 890 CRISPR screens in 629 cell lines, each manually curated and annotated from ...feasibility and power of CRISPR-based library screens in cell lines from humans and other species. By accurately... screens performed in 26 different breast cancer cell lines. Screenshot updated 2024. Each screen ...screen is captured by BioGRID curators, including the cell line/tissue type, enzyme system, sgRNA library and...example phenotype screens with read-outs other than cell viability. The following video provides an in-depth...

...six different research constellations: Doench/Root (2), Zhang (3), Wu (4), Yusa (5), Wang/Lander/Sabatini...Bioinformatics 33.7 (2016): 1099-1100. PubMed PMID: 28414855. 2. Doench, John G., et al. "Optimized sgRNA design to... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...more than 8x107 successfully targeted cells. Depending on the cells used and the readout, this could be ...My group works on immune cells in different contexts. Most of the immune cells we study are migratory, ...CRISPR modified cells are transferred into an animal and the behavior of the transferred cells is followed...with an easily measureable cellular phenotype (e.g. survival of a cancer cell as a cytotoxic drug is added...

...closer to the clinic (Tsai and Joung 2016). Table 2: Unbiased Genotyping Options Technique Detection...PMID: 23792628. PubMed Central PMCID: PMC3773023. 2. Vouillot, Léna, Aurore Thélie, and Nicolas Pollet...Digenome Seq 0.1% Cell-free (in vitro) Works across all cell types Must be verified with cell-based method ...Requires large cell population, senstive to time since cell fixing HTGTS Not reported Cell-based Identifies...method IDLV 1% Cell-based Programmable, can detect DSBs in live cells Not as senstive as other unbiased methods... use single cell clones for in vitro CRISPR experiments. The likelihood that a single cell clone derived...picture of the impact of CRISPR editing in the model cell line or organism. Several options exist for unbiased...

...Genome Editing and The CRISPR Software Matchmaker Day 2: Homology directed repair: CRISPR 101: Homology Directed...predict that we’d be able to edit RNA in mammalian cells with CRISPR? I certainly didn’t, but now we can ...updates We hope you enjoyed the #12DaysofCRISPR, a celebration of our updated blog resources. Not on social ...Improve HDR Efficiency for CRISPR Editing in Human Cells Day 3: Cpf1: Cpf1: A New Tool for CRISPR Genome ...

...fusion location, there are many choices to make. 2. Prime Editing: Adding Precision and Flexibility to...introduction of nucleic acids into bacteria or eukaryotic cells is a common technique in molecular biology and scientific...

...affinity and specificity. Trends in Immunology, 29(2), 91–97. https://doi.org/10.1016/j.it.2007.11.004 ...animation.)  Many types of immune cells, from mast cells to B cells, express Fc receptors that bind to...) antibody-dependent cellular cytotoxicity (ADCC) c) antibody-dependent cellular phagocytosis (ADCP). ...opened in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive when holes...die.  Antibody dependent cellular cytotoxicity (ADCC) Antibody dependent cellular cytotoxicity (ADCC) is...(NK) cells expressing Fcγ receptors binds to IgG and release cytotoxins that kill the target cell. If ...response, by binding to Fc receptors on other immune cells. (psst! Need a quick review of the different immune...