...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. ...

...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...

... 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...