...and understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification...bacteria and nearly 100% in archaea, though Class 1 remains largely underutilized compared to Class 2. Class...effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous grasp... genes, Cas1 and Cas2, which are critical for inserting short fragments of foreign DNA into the CRISPR...CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems called...a CRISPR-associated transposase (CAST) that can insert large fragments of DNA. The V-F variants, which...discovery algorithms find new candidates for CRISPR proteins. A type VII is already on the horizon (Altae-Tran...

...recombination is the process by which nearly all domains of life repair genomic damage, specifically double...sequence homologous to the chromosomal site of insertion. The linear PCR product is introduced into the...the cell by direct transformation. A given insert will typically contain both a protein modification sequence...imaging studies rely on direct fusion of fluorescent proteins (FPs) to a yeast gene of interest. These fluorescently...abundance, movement, and localization of individual proteins, but also investigate protein-protein interactions...interest, allowing for easy capture and detection of proteins and complexes, without the artifacts sometimes... PCR product from this collection will yield an insert with the format (protease site)-6xGly linker-(epitope...

...limited to traditional restriction enzyme cloning. Instead, you can choose a molecular cloning technique that...takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction... inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined...vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO cloning adds...Method B to create an entry clone: TOPO cloning the insert into an attL-containing entry vector.   Method...interest and a attL-entry vector. This fragment is inserted in a multiple cloning site (MCS) of an attL-containing... create an entry clone: Restriction cloning the inset into an attL-containing entry vector.   Pro...

...co-repressor important in development. Groucho family proteins are found in flies, worms, frogs, mice, and even...the Shivdasani Lab from the Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic...named by a postdoc in Cliff Tabin’s lab who took inspiration from a comic book his daughter was reading. SHH...synthetic transposon system that can be used to insert specific pieces of DNA into vertebrate chromosomes... et al., 1991, Jordan and Karess, 1997), which contains the regulatory regions that are necessary for ...spaghetti? I actually couldn’t find the specific origins of the name but some of our Addgenies had an idea...plasmid in our repository, plasmid 39624, that contains cmr.   tinman and bagpipe, two genes important...

...split protein like Cas9 is to use split inteins. Split inteins are a pair of naturally occuring polypeptides...toxicity. More AAV-based CRISPR systems Smaller Cas proteins SaCas9 isn't the only CRISPR enzyme that’s small...advantage that it can be turned off by anti-CRISPR proteins. The Sontheimer lab used NmeCas9 with AAV to edit...disease-causing loci in mouse liver.  Two other smaller Cas proteins include Cas12b and CasX. While there are no papers...papers yet published that deliver these Cas proteins with AAV, at just 1,108 and 986 amino acids, respectively...polypeptides that, when at the ends of two proteins, mediates protein trans-splicing, similar to an intron...developed a split intein spCas9-AAV toolbox that retains the gene-targeting capabilities of full-length ...

...upstream of either NGA or NGC PAM sequences (Kleinstiver et al. 2015). From these screens, they identified...mutations found in the REC2, REC3, and PAM interacting domains and allows for expanded PAM recognition as well...off-target activity (Hu et al., 2018).  In 2020, the Kleinstiver lab reported the development of near-PAMless ...the CRISPR toolbox The isolation of novel CRISPR proteins has and will continue to dramatically increase...which makes it easier to target AT-rich genomes (Kleinstiver et al., 2016). Type VI CRISPR systems, which ...Wolfe SA, Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome... plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng...

...cleave affinity tags from purified recombinant proteins at small and large scale.  This new technique ...technique involves selection in the cytosol (instead of on the yeast extracellular surface) which enables tracking...demonstrated to work for evolution of other target proteins and might be useful for your studies.  Find the... Angela Abitua Genetically encoded fluorescent proteins are often used to label  specific structures, ...within cells. However, sometimes these fluorescent proteins might not localize to the right places, which ...to improve nuclear localization of fluorescent proteins, they found that adding a triple nuclear localization...in the cytosol of E. coli, however the nanobody proteins tend to get stuck in inclusion bodies. Takayuki...

...you produce viral vectors using viral envelope proteins from another virus to either restrict or broaden... but you use a different envelope glycoprotein instead of the wild-type glycoprotein. The pseudotyped ...protein is encoded on the envelope plasmid which contains a promoter, the envelope gene, and a polyA tail...virus:  Lowering cytotoxicity. Different envelope proteins have different levels of cytotoxicity and the ...pseudotyping methods with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details, check...in serum (DePolo et al., 2000). Other envelope proteins for pseudotyping While VSV-G is the most commonly...for pseudotyping, there are many more envelope proteins that can be used for specific purposes as described...

...Strong insect promoter from baculovirus Constitutive  Commonly used in expression systems for insect cells...to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which is necessary...responsible for initiating the transcription of your insert into RNA. In practice, the term "promoter" describes...regulatory factors: enhancers, boundary elements, insulators, and silencers; however, some "leakiness" of ...Strong hybrid mammalian promoter Constitutive  Contains CMV enhancer, chicken beta actin promoter, and...with Tetracyline or its derivatives.  Typically contains a minimal promoter with low basal activity and... promoter. Ac5 General expression mRNA Strong insect promoter from Drosophila Actin 5c gene Constitutive...

...                           California Institute of Technology             Johns Hopkins University                           ...                           National Institute for Physiological Sciences Broad Institute                                       ...for tagged and untagged expression of SARS-CoV-2 proteins in bacteria, yeast, and mammalian cells. We teamed...from the Michael J. Fox Foundation are used in Parkinson’s research.  The extensive resource from the Structural... determine the three dimensional structures of proteins.  The RESOLUTE Consortium collection enables research... collection, a ‘One-Stop Shop’ for Fluorescent Proteins, and plasmids from several labs at Florida State...

...problems? Instead of directly fusing a fluorescent protein to your protein of interest, you instead fuse it...possibility of long-term imaging studies. If you instead overexpress your protein at higher levels, you ...is at a very high concentration. Overexpressed proteins also have the potential to form aggregates and...to the synthetic SunTag scaffold. This scaffold contains 10-24 copies of the short epitope GCN4. GCN4 recruits...SunTag, they attempted single-molecule imaging deep inside the cell, in the nucleus and cytoplasm. Again, ... of SunTag compared to traditional fluorescent proteins Improved brightness and signal-to-noise ratio... fluorescent proteins...

...alarming results are due, at least in part, to insufficient and non-standard record keeping despite scientist...to capture ideas, write down certain notes or brainstorm, but we have to admit that paper-based record...functionalities that are hard to imagine on paper, such as instant search through all saved data and automated writing...maintenance and support.” Besides that, academic institutions tend to think about the long term, so they need... once they establish the system within their institution. In the study, respondents were also asked about...providers used to supplement their paper notebooks. Instead, all of the important information is consolidated... In addition, if you are working for a large institution or company, site licenses might be available ...

...please use one of these methods to ensure your insert retains the 3' A overhang: Use a mixture of proofreading...below, the “A” overhang on the blue PCR product insert comes from using Taq polymerase for the amplification...these kits also often have the topoisomerase site inserted into a beta-galactosidase cassette allowing a ...clones. Once you introduce your 3’-end “A” overhang insert, the magic of TOPO cloning happens. Basic procedure... Colonies: You can confirm the presence of your insert by PCR, restriction digest, or sequencing. Pro ...concentration or you are cloning an extremely large insert. Since the standard ligation reaction is fairly...PCR Use REPLACR Mutatgenesis to Easily Generate Insertion and Deletions in a Plasmids Learn How to Verify...

...own machinery to replicate the viral genome and proteins. This is, for the intrepid researcher looking ...Virus structure While all viruses have a genome inside a protein shell known as a capsid, some also have...can either follow central dogma (gDNA to RNA to proteins) or require an intermediary DNA replication step...transcribed into mRNA, directly translated into proteins, or, in the case of retroviruses, reverse transcribe...transcribed into mRNA before being translated into proteins. RNA viruses replicate their genomes using a variety...egg sac. (Okay, some enveloped viruses bud out, instead of lysing, but they do generally still kill the...replicative ability. The remaining particle, which retains the protein coat (and envelope), has a “gutted”...

...Plasmids 101: E. coli Strains for Protein Production Addgenie Julian Taylor-Parker explains why you might want...Homology Directed Repair Addgenie Chari Cortez explains some of the basic science behind homology directed...production and gives a quick rundown of some of the strains you can use to express your protein of interest...find a successful clone? Addgenie Jessica Welch explains how Blue-White screening could be a potential ...Addgene Science Communications Intern Mary Gearing explains how Cas9 homolog, Cpf1, may make it easier to ... able to help you. Head Addgenie Joanne Kamens explains what career coaches do and offers up some career... to use with CRISPR as the cargo. Mary Gearing explains some of the clever ways to successfully combine...

...Oliver Griesbeck of the Max Planck Institute for Neurobiology has been working on genetically encoded...used for ratiometric in vivo imaging. Griesbeck explains, while these Twitch sensors are always bright,...particular advantage for observing events taking place inside cells that are on the move, such as T lymphocytes...Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular imaging of... should require the simple swapping of binding domains, in practice optimizing the sensors involves plenty...solution: they devised self-associating fluorescent domains whose association is disrupted in the presence ... of mOrange2 and mCherry as donor and acceptor domains. He says, these new colors might now enable observation...

...Negative control vector for pMKO.1 puro; Contains shRNA against GFP William Hahn 10676 pMKO.1 GFP Derivative...expression; Recommended for cloning new shRNAs (contains a 1.9 kb stuffer region to easily visualize products... Negative control vector containing non-hairpin insert David Root 1864 pLKO.1 ‐ scrambled shRNA Negative... (e.g. pSUPER) can be cloned into this vector; Contains a 5'LTR and should be packaged using the 2nd generation...Derivative of pMKO.1 puro with GFP instead of puromycin resistance gene William Hahn Additional Resources...cells and transgenic mice by RNA interference. Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang...

...blog Read our fluorescent proteins blog posts Download the fluorescent proteins 101 eBook Read our plasmids...cascade of caspases, protease enzymes that cleave proteins. Executioner caspases are activated last in the...Zhang et al., 2019). How does FlipGFP work? GFP contains 11 β-strands and one central ɑ-helix that can ...10 (β10), and (3) β-strand 11 (β11). Each part contains components and residues that are necessary for...specific protease that is commonly used to cleave proteins. This protease reporter based on sfCherry however...in FlipGFP can be applied to other fluorescent proteins to create an array of multicolor reporters that...fluorescent protein fusions Browse the fluorescent proteins plasmid kits ...

...pMinDisplay vector. GPI contains a C-terminal glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane...update our iGluSnFR post! Here, we look at the origins of the system and explore advances that have happened...happened since our original 2018 blog post. The origins of iGluSnFR   iGluSnFR was developed in 2013 by...an increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing...localization through different C-terminal anchoring domains The original iGluSnFR in the pMinDisplay backbone...introduce iGluSnFR3 in two additional anchoring domains – GPI and SGZ. Fig: 4 Sequence schematics...Fluorescent Protein Biosensors Browse All Fluorescent Proteins Find AAV Tools References Marvin, Jonathan S....

...structural component of atherogenic lipoproteins, shuttles lipoproteins through the circulatory systems, ...LipoGlo: A reporter for measuring ApoB bound lipoproteins Article contributed by Leah Schwiesow A leading...cardiovascular disease is driven by atherogenic lipoproteins that accumulate within the vascular wall. ApoB...formation of atherosclerotic plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their... Steven Farber and colleagues at the Carnegie Institute for Science have developed the LipoGlo system ...created an endogenous ApoB-NanoLuc reporter by inserting the NanoLuc luciferase downstream of the apoB ...requires binding of bacterial enhancer binding proteins (bEBPs) at a sequence upstream of the promoter...