...drastic shift won’t happen overnight, but there are a few reasons why you might choose NgAgo over CRISPR...TtAgo-mediated cleavage in mammalian cells, likely due to high temperature requirements. To solve this problem,...preclude future HDR, which could lower HDR efficiency. Higher sensitivity to off-targets More research is needed...other small nuclear DNAs from binding NgAgo, it might make it difficult to use NgAgo to disrupt multiple...Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants Browse All Genome Engineering...

...explains, while these Twitch sensors are always bright, they change color as they shift from "off" to ...They identified a toadfish TnC variant which had a high-affinity for calcium binding and developed a version...see tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes,...and acceptor domains. He says, these new colors might now enable observation of the interaction between...Addgene? Check out our Hot Plasmid page, where we highlight some of the popular & newly available plasmids...

... sections of scientific articles (gasp). To my delight, not only was I able to make the edits without ...all about the MOI. In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively...robust cell line. If your cells are sensitive, it might be more difficult to get them to grow out. The only...presence of the band, you could run another gel that highlights the important results (for example, if you needed...a human cell line with a gene knockout was straightforward, and is a testament to how far genome engineering...David Root and is described in Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation...

...reporter that has a large dynamic range and high brightness. The Shu Lab from UCSF designed an improved...reporter that has a large dynamic range and high brightness. This reporter achieves approximately 100-fold...immune system, and cell turnover. Apoptosis is a highly controlled process that is triggered by internal...a TEV protease cleavage site. TEV protease is a highly specific protease that is commonly used to cleave...

...butterflies in your stomach, the virtual approach might be less stressful. For both conferences, there was...Bachle. (For presenters, there’s also less stage fright!)And depending on the conference, the audience ...can upvote a question and the host will see the highly voted questions,” says Zhang. “Usually the moderator...moderator starts by asking the speaker the highly upvoted question.”If the Q&A session ran out of time, ...for Everyone.  While this type of interaction might not work when you are giving a research talk, there...difficult in the virtual format than in-person. You might miss out on the short chats between sessions or ...

...match your needs. If YFG is thrillingly new, you might have to make a transgenic fly in order to study ...study it. The process is fairly straightforward. Basically, you place your gene of interest in a plasmid and... trying to overexpress or knockdown a gene, you might need different plasmids. Harvard’s DRSC/TRiP Functional...gene to select for transformed bacteria, but you might be wondering, “How do I know which of my flies have...integration will have eyes that are anywhere from light yellow to red. The variation in color depends on...strategy and the services offered by the company, you might have to map into which chromosome the transgene ...

...neuroscience novice (newb). In this post, I’d like to highlight some of the things that popped out to me as a ...novice and indicate why other neuroscience newbies might want to get into the field. Find Optogenetics Plasmids... neuroscience but are afraid you don’t have the right kind of biological expertise to get into the field...’ve developed in your current work to make new insights in neuroscience. Even if you're currently a synthetic...interested in using AAVs because they provide a highly versatile means of delivering genes to many different...

...is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works by using...exploring these interactions, and can give valuable insight into DNA expression and protein interactions, including...require crosslinking, which uses formaldehyde or UV light to create strong but reversible bonds. This will...options for this step are sonication, which uses high-frequency sound waves to break up the DNA, or enzymatic...attach the proteins to the DNA, depending on how tightly the proteins bind natively. But the amount of cross-linking...few rounds of adjustments, to get everything just right!  Fig. 2: Goldilocks is actually celebrating...

...potential for microbial contamination. Additionally, the high activity of these enzymes implies that even a minute...a protein of interest). Mesophilic microbes are highly preferable (and successful) as host systems since...coli expressing the chaperones protein resulted in higher levels of growth compared to the E. coli strain...an outreach scientist from Addgene. The talk highlighted the importance of reagent repositories and the...leveraged for harmonious progress.     We’re always delighted to engage with potential collaborators and interested...

... activation and knew she was on the right track. The next insight was to assemble a synthetic transcription...bioengineering expertise? “This is the next step in high-throughput, genome-wide screening,” Konermann said...sgRNA library. Our SAM-mediated screens exhibited a high degree of consistency and validation with >80% effectively...software for analyzing the data. It should be straightforward to apply the new library for use in various...an engineered CRISPR-Cas9 complex. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...

...microscope where the light source is mounted above (epi) the specimen and the excitation light passes through...cause a higher-energy electronic transition in a fluorescent molecule (see figure to the right). The excitation... proteins are excited by 750 nm to 760 nm laser light, enabling dual color imaging studies with blue or...fluorescent proteins (proteins that fluoresce after a light-induced chemical reaction) or photoconvertible Keade... Keade proteins (protein that undergoes light-induced irreversible photoconversion from green fluorescence...

...recommendations that postdoc salaries be raised to $50,000 - higher than the level being raised to now - across a wide...from postdocs and faculty (at time of writing, Brigham And Women's in Boston also just announced that ...before the injunction, are raising salaries, even in light of the injunction. In addition, PIs at some of the...Institute, where salaries had already been raised higher previously and all 6,000+ postdocs at the University...contract - negotiated by their union - securing higher salaries. This decision will carry over into other...information, many assumed that those living in areas with high costs of living would get much better salary increases...

... experience, we found that fluorescent proteins might not always be the best option. Fluorescent proteins...antibodies coupled to nanogold particles, that allow a high-spatial resolution of the protein localization. ...hypothesized that a different tag fused to the hM4Di might improve the transport of this DREADD to the plasma...even when fused to mCherry. But our data does highlight the importance of a careful characterization of... Proteins 101: GFP Fusion Proteins – Making the Right Connection.     Thank you to our guest blogger...

...market because of life-threatening health effects. Eight of these posed greater health risks for women than...cause human suffering and death. Doing research right has the potential to save lives and money. A 2014...innovations It is crucially important to get the research right from the beginning. This is the goal of Gendered...reasons why drugs fail - sex is one of them. Why might the sex of the cell be relevant? Research shows ...disparities in disease severity. Such disparities might suggest underlying sex-based influences on physiologic...curricula. Corporations need to integrate these insights into product design. But eyes have been opened—and...

... mind, when planning an experiment users should weigh the benefits of enhanced biosafety versus a potential...Tet-off transcriptional activation system to drive high levels of expression of certain viral components...manufacturer these changes result in yields up to 25x higher than other available systems. Producing lentivirus...obstacle in obtaining usable lentivirus is producing a high enough concentration, or titer, for your experiment...a double harvest approach at 48 and 72h produces high titer virus for transfer plasmids with small inserts...11589831. Jiang W. et. al.. An optimized method for high-titer lentivirus preparations without ultracentrifugation...

...Although methods to switch on Cas activity using light or drugs have been developed, the field lacked an...anti-CRISPR proteins work? Anti-CRISPRs proteins are highly diverse but most block CRISPR activity one of three...Regulating Acr proteins by inducible promoters, light, or small molecules could achieve rapid, dynamic...allows anti-CRISPR activity to be controlled by blue light (Bubeck et al., 2018). 3. Reduce cytotoxicity of...pathogens possess CRISPR-Cas systems, phage therapy might not be as effective. This is where Acr proteins ...CRISPR Plasmids for Your Research Interested in light-inducible Cas9 activity? Check out these these plasmids...

...overexpression construct is being delivered, that is a higher risk cargo for a researcher to come into contact...essential for cellular function, this would also be a higher risk vector. While it is unlikely that you, or ...other BSL-2 materials, you’ll need to use aerosol-tight caps, compatible with your centrifugation speed,...useful. Some organizations or cities may require a higher percentage of the decontaminate to be used for ...researchers. Note that safety policies will vary slightly from organization to organization, so you’ll need...cut a few corners. For many researchers, viruses might be the most dangerous things they work with, and...

...very powerful for high level recombinant protein expression. These systems enable high expression of very...biology research (eg. polymerases and other enzymes might be purified and used to manipulate DNA), or in medicine... through stable cell lines. Both methods produce high protein yields if transfection is successful. Some...if a desired protein product is toxic to cells at high concentrations. Despite their advantages, mammalian... promoter and induced by lactose, provides the highest level of protein expression. Despite their ease...

...Association for Cancer Research Annual Meeting, you might consider including more background on how your virus...to expertly show off your science.  Prepare the night before: To avoid rushing out the door without your... tack up next to your poster. Arrive early: You might be nervous before your poster session, or it may...walk up to someone’s poster, and then he/she dives right into the background without an introduction. If ... in his/her work, and would enjoy hearing the highlights so that you can appreciate the impact of the ...

... use to introduce yourself and what you do. You might want to include your university and your advisor...new at a conference, one of the first things you might want to know is what their project is. Or maybe ...familiar with. You can tweak your elevator pitch slightly if you know a bit more about the other person....elevator pitch several times at the conference, you might notice some trends. Were there parts of your elevator... up on the board and meet your poster session neighbors. During the session, if someone comes up to my...and your resume and prepare business cards. You might not consider being a student or postdoc a “business...

...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...Plasmids and Resources page.  A new system uses light to deactivate photocleavable gRNAs within seconds... assembly of CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. ...

...Every few months, we highlight some of the new plasmids, antibodies, and viral preps in the repository...nucleocapsid RNA chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene ...rely on synthetic dye molecules, which are much brighter and more photostable than fluorescent proteins...Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation in the...illumination regimes with fast activation kinetics and high responsivity near the resting membrane potential...

.../s12915-019-0682-0. Beck, C., Gong, Y. A high-speed, bright, red fluorescent voltage sensor to detect...the kinetics of voltage sensing to signal output slightly slower than other approaches. Functionally, the...photobleaching, lack of red-shifted indicators, and brightness of fluorescence all have room for improvement...transport, not fluorescence, they are not nearly as bright as the other GEVI systems that use engineered FPs...one component counterpart and is significantly brighter and available with red-shifted fluorophores (find...

... trying to perform high-throughput experiments. If a user wants to carry out a high-throughput genetic...the DSB. CRISPR has allowed scientists to tag and light up endogenous genes of interest to better understand...convenient one tube assembly reaction to generate high-throughput targeting vectors. These vectors can ...fluorescently-tagged proteins at endogenous loci, shedding light on previously unknown protein functions. References.... and Eric M. Jorgensen. “SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis...

...Kits can also have the annoying tendency to runout right when you need them and to accumulate a bunch of ...precipitated, dried, and then rehydrated. There are some slight modifications depending on the type of DNA you’... not bind silica or glass, but the addition of a high concentration of a chaotropic salt (guanidine hydrochloride...: agarose gel slices Product: DNA fragments You might be wondering, “How is milk going to help me extract...purify DNA ranging in size from 100 bp to 48 kb with high yields and without degradation during the extraction...nucleic acids. Containinants bind cellulose less tightly than nucleic acids, so placing the dipstick into...

...making the purification process simpler. Due to its high yields, E. coli is often used for the production...transfection, depending on whether the heavy and light chain genes are contained to one plasmid or if they...on an SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside your ladder and ...software. Any bands on the gel beside the heavy and light chains can also be measured. Contaminating bands...Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production...capture process based on magnetic beads from very high cell density suspension. Biotechnol Bioeng. 2021...

...). I’ve assembled a series of screenshots to highlight changes to both the design and functionality of...involves delivering a calcium biosensor using AAV, you might decide you’d really prefer to order ready-to-use...look at filters in a moment. First, I want to highlight the convenient ways our new search tool lets you...you know the ID number for a plasmid, your straightforward search is now even easier with a direct link...

...average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains they were...were derived from. They also transduced a higher number of neurons overall (GFP+ cells that also stained...dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are labeled..., or blue (RGB) fluorescent protein (4a-c). When high doses of this viral cocktail are injected, by chance.... tet-off transactivator (tTA), is injected at a high dose to maximize the number of cells that are labeled...part system, color diversity is maintained at both high (figure 4e) and low doses (figure 4f) of the tTA...

... information about insert orientation, then you might consider designing orientation-specific primers....primers. Blunt end cloning is an example of when you might want to know the orientation of the insert. One member...with this and a few other colony PCR tips are highlighted below. Preparing template: Pick a single colony... liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you pick and only...size. Make sure to use an appropriate molecular weight standard for reference and to add a loading dye...

...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved...alternative to genome-wide RNAi screens, which although highly effective, are affected by low on-target efficacy...unclear if off-target cuts confound the results of high-throughput CRISPR screens. Do short guides (17-...mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target activity. Short... with only a small loss of on-target activity in high-throughput screens. The human and mouse versions...measurement of off-target activity using Cas9 toxicity in high-throughput screens." Nature communications 8 (2017...

... advice on areas where you might need some help. Today, we’re highlighting an oft-underlooked transferable...skate as a starting point, their ideas scored 37% higher in originality. It may not be easy to optimize ...truly disagrees with the group (because they will fight more adamantly for their solution), selecting a ...power to change anything. You may not have the highest position or the longest tenure, but don’t let that...

... confirmed that indeed the gRNA-pMB1 had a slightly higher leaky expression in BL21(DE3) that resulted...customizable cargo gene in the form of RE-cargo-LE (RE, right end; LE, left end) pTnsABC expresses the transposases...glucose dehydrogenase expression that is 2.6 times higher when compared to the industrial strain. In our ...copies of integrated genes. We obtained a stable and high-yielding strain which had 6 times the yield of the...transfer on transposition-inducing agar lead to higher cargo copy numbers. (D) The effect of gene dosage...

...James from Binning Singletons. During Junior High and High school in the 80s and 90s, Joe was that kid ...asked if she had a few minutes to point me in the right direction for my future work. She graciously took...match people within similar fields (ASM Microbe has eight tracks), something we didn’t have enough people ... ASM Microbe, we connected people who otherwise might never have met. Binningsingletons.com will be the... but before going to graduate school, Joe taught high school chemistry for a year. The teaching/mentoring...

...our plasmid lab to develop a reliable and robust high quality viral vector service, making an array of...protocols we use in our lab — being able to order high quality viral vectors from the Addgene repository...on quickly in the scientific community, turning slightly under ten requests a day in 2017 to over fifty...that are activated, respectively, by substrates, light, or engineered ligands (Figure 2). Biosensors, which...our own repository data, giving us significant insight into which vectors are likely to be of most use...plasmid repository gave us the experience to launch a high-quality ready-to-use viral vector service. But in...

...chance of cross-reactivity Sandwich High specificityHigh sensitivity Longer protocolIncreased chance...several drawbacks. Direct ELISAs generally have higher levels of background staining. Antigen-containing...simple protocolReduced chance of cross-reactivity Higher level of background stainingLess sensitivityLess...availability of conjugated antibodies Indirect High sensitivityGood antibody availability Longer protocolLess...Antibodies 101: ELISAsAntibodies 101: Selecting the Right Antibody Antibodies 101: Polyclonal Antibodies Resources...

...which cuts at NRN PAMs with high efficiency, as well as NYN, but with a slightly lower efficiency. Information...available, it can be difficult to decipher which one is right for your unique experiment. Here, we will introduce...the genome and consider using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing...location and type of edit you need to introduce highly specific? Perhaps you need to introduce a single...choose from within the Cas family if Cas9 isn’t right for your experiment. Keep an eye out for our upcoming...

...up-regulated. For instance, generally, the more highly expressed a gene is under native conditions, the... achieve using a CRISPRa. Your gene of interest might already be hitting an upper bound of activation ...scale CRISPR-based screens can be used to gain insight into protein structure and function (Pan et al...., Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming...doi.org/10.1016/j.cell.2014.09.029 Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...j.cels.2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous...

...Pitfalls of Fluorescent Protein Fusions Observing a bright signal for the first time after cloning an FP fusion...studies and those of others (5, 6, 7, 10, 11) highlight the very real consequences of misfolded dark FP... blue or cyan and yellow moxFPs. The inherent brightness and spectral characteristics of the moxFPs are...standard FPs. The increase in signal is not due to a brighter FP, but rather because of the correct folding ...listed above. In addition, because the moxFPs are highly monomeric, they are an excellent choice for fusions...

...reported in PLOS publications. The policy was not an overnight revelation, rather it was the result of consultations...A distinct (perhaps similarly impoverished lab) might apply different or novel tools and assumptions to...It could be argued that data generators may, in light of the policy, hold onto data longer before publishing...include suggests pragmatic adoption (although it might be argued various authors have simply chosen to ...significant effort. Our standards of data recording are highly variable and usually left to individual researchers...contact more senior colleagues. Secondly, science is highly technical and this has a natural, albeit unintentional...

...contract citizens enter into in order to protect their rights (2). Less formally, governments are the organizations...public will lose trust in scientists if the march highlights data supporting unwanted results (14). Some loss...surrounding a matter or policy should have more weight than how any one person feels about it. And yet...evidence’ on Trump’s wiretap claim The Chronicle of Higher Education: “March for Science” Organizer Says It...Science Foundation: Universities Report Fourth Straight Year of Declining Federal R&D Funding in FY...

...Immunofluorescence (IF), is an immunoassay that brings to light the cellular world. The technique allows you to ...indicates your target’s location. Some other terms you might see associated with IF are immunocytochemistry (... of other antibodies. But, once you do find the right protocol, multiplex IF can give you impactful and...sample on slides, and confocal   Figure 2: High-level overview of an indirect immunofluorescence...ones? Choosing a secondary is often much more straightforward than choosing a primary, so we’ll start there...the future.  We hope that this post has shed some light on immunofluorescence. The technique can provide...

...kDa single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody...minimizing oligomerization of the scFv. While you might assume that the variable domains of an scFv would...lower affinities, lower long-term stability, and a higher likelihood to aggregate due to their small size...library with plate-bound antigen helps select for high affinity scFvs. Bound phage are then proliferated...with plate-bound antigen to select for scFvs with high affinity binding. Bound phage are then proliferated... since direction injection to the eye results in high drug concentrations in the eye with minimal systemic...

...their regenerative abilities is expected to shed light on human aging pathological developments (Vieira...amphibians in science with early research providing insight into some of the most significant and fundamental... Image from Holger Krisp. The frog genome has high synteny with that of humans so they are often used...the world. With its extensive scientific history, highly characterized genome, and widespread utilization...produces many seeds), self fertilizing, a small yet highly characterized genome, and is easily maintained ...Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493–521 ...

...Every few months we highlight some of the new plasmids, antibodies, and viral preps in our repository...microscopy (cryo-EM) protocols typically require high concentration and purity of the target molecule,...or hundreds of particles surrounding each of the highly-visible beads on the cryo-EM grid.     ...denaturated (left) or complexes may dissociate  (right). B) Protein samples protected from AWI by helical...authors to determine structures at comparable or higher resolution than using more complex protective methods...

...heterodimers composed of one of 18 alpha and one of eight beta subunits. Both subunits have short intracellular...ectodomains can extend and open, binding ligands with high affinity. This extended-open state is key to strengthening...flexibility is crucial for their function. With the right research reagents, researchers can measure this ...developed and defined. According to the Springer lab, a highly specific function-blocking antibody, αVβ3.13, may...as new reproducible rat or mouse rAbs. The other eight are chimeric antibodies composed of rat or mouse... recently showed RGD-binding integrins demanded higher tensions before they would enable cells to spread...

... To better highlight the great content contributed by our bloggers each and every month, we've decided...Cpf1 Update: Comparison to Cas9 and NgAgo As you might expect given the excitement surrounding anything...month is from guest blogger James D. Fessenden at Brigham and Women's Hospital. This post describes how to... our posts if you have any questions or useful insights. If you're interested in blogging for us, fill...

...fortunate that there’s a guide that directs cleavage right before the stop codon!  Figure 3: How to ...guide sequences (arrows) and their PAM (bold highlighted text) and cleavage sites (slashes) are shown....plasmid options for templates - see the example at right using blue flame plasmid pmScarlet_C1! After creating...primers to add T7 promoter-left homology arm and right homology arm to the desired insert (e.g., mScarlet...the main drawback of i-GONAD is that it requires a high-concentration, column-purified DNA template.   ...

...fixed sample. Depending on the sample type, this might be frozen or formalin-fixed, paraffin-embedded tissue...Depending on your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B).... Depending on the goal of your experiment, you might need to quantify the intensity of the different ...spatial relationship between the targets. Or, it might be sufficient to describe or classify the labeled...label a sample that expresses your target(s) at a high level, either naturally or by overexpression, for...similar between the two experiments. If not, it might be that the other antibodies are disrupting the ...

...Every few months we highlight some of the new plasmids, antibodies, and viral preps in the repository...deactivation). Both have significantly improved brightness, kinetics, and sensitivity. iGluSnFR4f is well-suited...and EGFP-expressing lentiviral backbones designed right here at Addgene. Here's what makes these new backbones...Protein Innovation (IPI) recently released a set of highly-specific recombinant antibodies against three of... — with efficiencies between 5% and 11%. It has higher efficiency and lower indel formation than methods...

...not having any confounding mutations, the risk is high. You are generating a gene therapy product with ...clinical trials. An off-target rate of 5% is far too high when considering human patients and the diseases...measurements at those sites to get an idea of how high the off-target frequency is (more on this below)...sequencing methods. Candidate site sequencing Sites with high sequence similarity to the gRNA can be sequenced...a DNA mutation, off-targets must be quantified slightly differently. For a comprehensive approach, RNA-seq...library screens, the same principle holds true - the higher the number of distinct editing events, the lower...