...to make sense of biology, with a special focus on gene editing techniques. References and resources References... versatile genome manipulation. Nature Reviews Genetics, 24(3), Article 3. https://doi.org/10.1038/s41576...DeepSpCas9, a deep learning–based model with high generalization performance. Science Advances, 5(11), eaax9249...). Chromatin context-dependent regulation and epigenetic manipulation of prime editing. Cell. https://...j.cell.2023.03.034 Resources More resources on the Addgene blog How to Design Your gRNA for CRISPR Genome ...and Flexibility to CRISPR Editing Resources on Addgene.org CRISPR: PRIME Edit Collection The CRISPR Guide...

...my students published their work identifying new genes required for stem and progenitor cell maintenance...incorporated a discovery-based lab on planarian regeneration into one of my own courses, I was excited to...be authentic – students should be attempting to generate new knowledge that will be of genuine interest...Provost’s course recently used a combination of phylogenetic analysis, structural prediction, and bench work...recombinant enzymes in bacteria, purified them to near homogeneity using a 6XHis tag, and compared their specific...

...from transient overexpression of protein fusions to gene-edited cells can decrease the number of fluorophores...Fluorescent Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids...in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion with your... dye ligand (e.g., FlAsH, ReAsH) and a short genetically encoded tetracysteine (Cys4) peptide tag could...TMP-tag) – These widely used systems consist of a genetically encoded enzyme variant tag that reacts specifically...such as paclitaxel. Unlike other systems, no genetically encoded tag is required as the small-molecule...strategies have trade-offs between the size of the genetically encoded tag, the speed and selectivity of the...

...contain either an ampicillin or kanamycin resistance gene. Every step along the way involves good aseptic ...of tube for each plasmid we have at Addgene. Each sample at Addgene has four layers of location which can...pages! Resources Avaliable on the Addgene Blog Read more Inside Addgene Posts! Learn all about Plasmids ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the... a microcentrifuge tube. Once a sample enters Addgene’s lab, a series of events is triggered. Each step...fluctuates throughout the year (see graph below). Addgene scientists inspect each sample and make sure they...takes over and prepares the sample for entry into Addgene’s repository. For long-term storage in our facilities...

... Ti plasmids in Agrobacterium tumefaciens. Genes & Genetic Systems 77:1–9 . https://doi.org/10.1266/ggs... Resources on Addgene.org Download the Plasmids 101 eBook  Browse plasmids at Addgene Read our Molecular...plasmid partitioning system in the bacteria, but generally it consists of a centromere-like region on the...Kamruzzaman et al., 2017). Additional resources on the Addgene blog Read all of our Plasmids 101 blog posts Learn...

...likely -/-, 50% are -/+, and 25% are +/+ for your gene of interest. Thus, to find a null clone, you should...edited. For many experiments, the end goal is to generate a clonal cell line, so it’s important to verify... for a needle in a haystack before going on to generate and screen individual clones. If a clonal line...al., NAR). This technique uses the trace file generated in Sanger sequencing and a simple algorithm to...with your sgRNA sequence. The TIDE software will generate a graph representing all insertions and deletions... 4 clones. Did you use a dual guide system to generate your knockout? Then you’re in luck! You don’t ... to visualize an edit. Most dual guide systems generate a deletion large enough that it can be visualized...

...IPTG. T7 RNAP is then available to transcribe the gene of interest from a T7 promoter on the plasmid. Many...::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein...cytosine within a particular sequence. Browse Addgene's curated list of Bacterial Expression Systems Table...DE3) F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) General protein expression BL21 (DE3) pLysS* Chloramphenicol...ompT lon hsdSB (rB- mB-) gal dcm rne131 (DE3) General expression; not recommended for toxic proteins ...lon hsdSB (rB- mB-) gal dcm araB::T7RNAP-tetA General protein expression  BLR (DE3) Tetracycline RecA-deficient...overexpression? The strains described above should generate sufficient expression levels for most purposes...

...mediated HDR) enables the tagging of endogenous genes close to native expression levels. However, it is...chimera are encoded by DNA. Since researchers can generate almost any DNA sequence in the way that they like...proteins is relatively straightforward. However, generating a fusion while keeping all of the native properties...challenging. In this blog I discuss strategies to generate fusion proteins and highlight some aspects of ... from Aequorea victoria and its variants are genetically encoded fluorescent probes. One of the limitations...restriction enzymes in the multiple cloning site for generating fusions. For the POI-fluorescent protein fusions...sufficient. For the fluorescent protein-POI fusions, we generate straight fusions, without any linker. When both...

...achieved through, for example, a previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside... on Addgene.org Ready-to-use recombinant antibodies at Addgene Addgene's Antibody Guide Addgene's Antibody... combines a marker with a signal. For clarity, Addgene uses 'labeling' to refer to antibodies and 'staining...Find streptavidin plasmids and biotin plasmids at Addgene!     Figure 1: Indirect detection of target...signal. Dump gating When you are working with heterogeneous cell mixes, like bulk lympho-/splenocytes, you...Antibody Plasmid Collection Resources on the Addgene blog Antibodies 101: Reading a Flow Plot Antibodies...

...do not naturally express the protein or have had gene expression knocked down or knocked out. If you are...antibodies Resources on Addgene.org Find written and video protocols Visit Addgene's antibodies collection...plasmid encoding your protein of interest from Addgene, transfected it into your target cells, now what...wet, semi-dry, and dry but all follow the same general principle where the gel and membrane are “sandwiched...may even find plasmids encoding antibodies from Addgene that you can make on your own.  To narrow your ... found this blog helpful be sure to check out Addgene’s blog for overviews of other antibody topics. ...10.4103/1947-2714.100998 Additional resources on the Addgene blog Find other tips and tricks for molecular ...

...which come from naturally-occurring immunoglobulin genes, recombinant antibodies are plasmid-based. Their... What is an Antibody? Resources on Addgene.org: Browse Addgene’s Antibody Plasmid Collection Find antibodies...cascades that clear these infections. Anything that generates an antibody response in the immune system is referred...in the DNA of the hybridoma can occur, known as genetic drift, meaning that over time, the antibody you...monoclonal antibodies, recombinant antibodies are a homogeneous population. However, unlike monoclonal antibodies...antigen binding, the variable regions alone can be generated as a fusion protein. An ScFv is made up of the...detect the presence of a single protein from a heterogeneous mixture - for example, cell lysate or media ...

... 18 lung cancer patients. This study used CRISPR gene editing to knockout PD-1 on immune cells harvested... Cell heterogeneity Tumors are composed of a wide variety of cell types. This cell heterogeneity is an...cells that escape the elimination stage acquire genetic alterations and alter cell-surface antigen production... promotes the production of VEGF to increase angiogenesis around the tumor. This in turn regulates the... immunotherapy. Using activated DCs ex vivo to generate cytotoxic effector T cells, and targeting specific...played a huge role. For instance, researchers are genetically altering tumor cells and immune cells to knockout...element of successful tumor progression. A tumor’s genetic composition is constantly in flux and is dependent...

...procedure and application to the major ALS/FTD disease gene C9ORF72. eLife, 8, e48363. https://doi.org/10.7554...101: Validation Resources on addgene.org Addgene's Antibody Guide Addgene's Antibody Protocols... outlined below. Knockout cell line approach Genetic knockout-based validation can conclusively prove...you are working with cells that are amenable to genetic knockout, this represents a very robust approach...to start with a screening procedure in easily genetically modified cell types (top). The specific approach...10.1038/s41598-019-55133-7 More resources from the Addgene blog Introduction to Gating in Flow Cytometry Antibodies...

...transition from weak, random binding to precision gene editing requires three major steps: RNA loading,...resources on addgene.org  Addgene's CRISPR Guide Addgene's CRISPR 101 eBook  Resources on the Addgene blog Components...from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo state...    Figure 3: Crystal structure of S. pyogenes Cas9 bound to guide RNA (PDB ID 4ZT0), shown opaque...sequence isn’t in place yet.     Figure 4: S. pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared... residues in the CTD can get cozy with DNA. S. pyogenes Cas9 recognizes the PAM sequence 5’-NGG-3’, where...complementary strand is mismatched.   Figure 5: S. pyogenes Cas9 bound to guide RNA and target DNA (PDB ID...

...of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation." Nature biotechnology (2014). PubMed...to select a tool based on your project and not general opinion. I could spend a lot of time running through... Online tools dominate the software space but generally rely on less powerful algorithms to detect off-targets...tools available mostly work on any computer but generally require working knowledge of the command line ...tools require some kind of data input in order to generate results. While different types of input are more...focus on specific organisms. For this reason I generally recommend that laboratories that have already ...PubMed Central PMCID: PMC4262738. Resources at Addgene Get Tips on How to Design a gRNA Read Our CRISPR...

...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...Over 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database...editing tools. We’ll summarize the major ones in Addgene’s repository later in the post. Advantages of prime...by double strand breaks has been widely used to generate precise changes. However, the efficiency of Cas9...Prime editing has a lot of promise for treating genetic diseases. In our summer Hot Plasmids post, we covered...demonstration of prime editing used to rescue a genetic disease phenotype in an animal model, which was...s41587-022-01527-4 Additional resources on the Addgene blog PRIDICT: Predicting Efficiencies of Prime...

...Resources on Addgene.org Addgene’s Viral Services Addgene’s Virus Protocols Addgene’s Chemogenetics Guide ...system.   Figure 2: General overview of chemigenetic tools. A genetically encoded component is delivered...Resources on the Addgene blog Viral Vectors 101: Chemogenetics Viral Vectors 101: Optogenetics Fluorescent... within a cell are genetically encoded voltage indicators (GEVIs) and chemigenetic sensors. These tools...on this later). Chemigenetic sensors take a hybrid approach and utilize both a genetic component as well... system. Single Fluor Chemigenetic Sensors This system consists of a genetically expressed enzyme, typically...about how viruses can be used for chemigenetics, biosensors, and generally as biological tools, our blog ...

...AAV1 Chemogenetics Scott Sternson New viral prep pAAV-CaMKIIa-hM3D(Gq)-mCherry AAV1 Chemogenetics Bryan...mCherry AAV1 Chemogenetics Bryan Roth New serotype pAAV-hSyn-hM3D(Gq)-mCherry AAV1 Chemogenetics Bryan Roth... Brian O'Neill.  More resources on the Addgene blog Addgene's Data Hub What is a Virus (and a Viral Vector...but browsing the 49 new preps available in the Addgene viral vector repository with a nice cup of hot ...Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor... serotype pAAV-hSyn-hM4D(Gi)-mCherry AAV1 Chemogenetics Bryan Roth New serotype pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato...prep pAAV_hSyn1-SIO-stGtACR2-FusionRed AAV5 Optogenetics Ofer Yizhar New serotype pAAV-CaMKIIa-ChRmine-oScarlet-KV...

...learn what Addgene can do to help. More resources on the Addgene blog Twenty Years of Addgene Sharing: ...researchers around the globe. Addgene’s educational resources As Addgene built out our scientific support...other videos on Addgene’s YouTube channel.   Figure 4: Global viewership of the Addgene YouTube channel...about virus, I learned from Addgene,” or “I learned to clone from the Addgene website.” We delight in seeing...When Addgene opened in 2004, we focused on creating a repository that made it easy to share plasmids,...accessible, due to a wide range of logistical barriers. Addgene worked to identify, reduce, and remove these logistical...accessible weren’t always the same thing, shaped both Addgene’s founding and our growth into the company we are...

...Community Addgene: In 2012 your lab first deposited plasmids with Addgene. In 2014, you joined Addgene's Advisory...lab. In June 2014, Dr. Fujii joined Addgene's Advisory Board.  Addgene: Your lab has worked extensively ... enChIP through Addgene. Retroviral Expression System for enChIP using CRISPR Addgene: Your lab recently...the plasmids of these systems to Addgene in the near future. Addgene: Has your lab been working on any...first knew Addgene around 2010 and found the scheme very nice at first sight in that Addgene takes care...ourselves without Addgene. So, when I received an invitation to join the Advisory Board of Addgene, I accepted...Hodaka Fujii lab's Addgene page and click the "Subscribe to Alerts" link in the Addgene Alerts box. References...