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...this need in the community, we approached it in the way Addgene approaches everything; we asked ourselves...some of the people and organizations who helped us reach this milestone. Viral service supporters We would...from laboratories all over the world. With this reach and exposure in the community, we saw the opportunity...shipping systems and protocols for viral vectors to reach scientists around the world. Our Software Engineering...our systems for this new type of material. The Outreach Team is getting the word out about this new resource...preps derived from plasmids in the repository. The Outreach Team has been making sure scientists know about...

...revolutionary physical approaches. However, with each new technical achievement, the complexity of the...disciplines. In biology, innovative new imaging approaches are constantly being developed which combine ...with the community are particularly successful. Machine-learning denoising of raw data (CARE), segmentation... greatly increases accessibility. Through this approach, the Planktoscope, a portable device for studying...and Deployment." bioRxiv. 2020. Diederich, B., Lachmann, R., Carlstedt, S. et al. A versatile and customizable...

...ions crosses the cell membrane, a threshold is reached and the neuron fires, sending the electrical signal...specificity far greater than the one that can be achieved using pharmacological or electrophysiological ...., 2016; Hudry and Vandenberghe, 2019). How to achieve strong opsin expression in specific neuronal cell...expression in vivo (Schnütgen et al., 2003). This is achieved through site-specific recombinases such as Cre...expression in specific neuronal cells? This can be achieved using Cre-dependent viruses, such as the optogenetic...love for educational science led her to pursue a teaching career, and she now is a very happy scientific...Philippe Coulon, and Ilka Diester. "Optogenetic approaches to study the mammalian brain." Current opinion...

... of each other. That is, multiple independent variables are assumed to not have any effect on each other...variables are affecting each other – or if you think they may be affecting each other – then a MANCOVA ...variance, data ranges. how closely data points fall to each other (see Fig. 1). Alpha values – numerical ... if only two of those samples are different from each other, or if every permutation of pairs within the...null hypotheses. Benjamini uses a complementary approach which identifies a p value cut off. This method...Duncan’s multiple range test, and several others. Each test has different likelihoods of returning false...

... Arnold’s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are...channelrhodopsin that’s successful in vivo. Using machine-learning to overcome channelrhodopsin design challenges...challenges, the Arnold and Gradinaru Labs developed a machine-learning model to help them construct “designer...packaged in PHP.eB, ChRgers and ChR2(H134R) both reach the brain, but individual neurons do not express... mice. A fiber-optic cable allows blue light to reach the brain. Created with BioRender.com. In the...implanted into the mice’s brains so that light could reach the VTA. Mice were then allowed to “self-stimulate...Robinson JE, Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin engineering enables...

...including difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away... sometimes incorporating chemically inducible approaches to provide temporal control, this new system,...Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid and reversible recruitment of...target multiple loci at once. In addition, the approach allows minute-by-minute analysis of the kinetics...transcription factor as well as reporters specific to each transcription factor. They then induced cultures...induction via estradiol and progesterone do not affect each other, these systems can be used together. Aranda-Diaz... gene expression regimes that cannot be easily achieved with classical gene overexpression and knockout...

...thanks to our guest bloggers Joachim Goedhart and Marieke Mastop! Joachim Goedhart is an assistant professor...This post was contributed by guest bloggers Joachim Goedhart and Marieke Mastop from the Section of Molecular...two posts in this series described a practical approach to selecting a bright fluorescent protein and ...homodimerization is characterized by an affinity of 110 µM (Zacharias et al, 2002), meaning that at this concentration...cells. To understand how well fluorescent proteins approach this ideal situation, cellular assays have been...fluorescent probes. You can follow him on twitter: @joachimgoedhart.   Marieke Mastop is a PhD candidate at the...

... identical in sequence to the unmodified enzyme. Each half of the base editor is fused to a fast-splicing...GFP. The lab tested the base editors in vivo and achieved therapeutically relevant editing efficiencies ...heart, and skeletal muscle. The dosing required to achieve these efficiencies was also between 1013 - 1014...organisms such as E. coli and yeast. Because this approach uses standardized parts, it allows for greater...controlled by red light by splitting Cre and fusing each half with PhyB or PIF6, phytochrome-like fusion ...prosthetic group PCB, PhyB and PIF6 dimerize, bringing each terminus of Cre together to restore Cre activity...fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene that...

...process termed hydrodynamic focusing. This allows each cell to pass through the path of the laser in single-file...fluorescent light sources. Visible light illuminates each cell as it passes through and scatters in different...to a voltage pulse, called an event, and assigns each event to a channel number based on intensity. Higher...experiment uses 2 or 3 different fluorescent colors each measuring a different target. Flow cytometers however...stain the intracellular target. When using this approach, avoid fixation-sensitive fluorochromes such as...charged as they pass through a metal deflection plate. Each droplet contains a single cell that is assessed ...collection vessels and can sort single cells into each well allowing users to easily isolate and subsequently...

...intended. Sharing unverified plasmids can lead to far reaching consequences, from wasting time and money on optimizing...Depositing scientists provide different types of data for each of their plasmids, from descriptions of key components...matter the level of information that is provided, each deposit goes through the same QC process to ensure...deposited sequences and plasmid information.  For each plasmid deposited at Addgene, QC scientists confirm...selectable markers, and fusion proteins listed for each plasmid  If the plasmids pass all inspection points...mutated; elements that are difficult to sequence; approaches to confirm plasmid sequences without a reference...confirm their presence with confidence. If this approach does not resolve the identified differences, the...

...need for low-cost, field-based diagnostics, our approach could make an impact. Our motivation was underscored.... We also were careful to set up clear tasks for each member of the team to accomplish so that many elements...expertise of the group and yet never hesitated to reach out to the greater research community when we needed...involved in the effort. This inclusive and open approach by all involved definitely contributed to our ...). Using toehold switch-based RNA sensors, our approach uses portions of the pathogen’s genome as unique...portable quantification of diagnostic results.  Our approach now detects samples at clinically relevant concentrations...longer-term aim is to advance our programmable approach to other critical needs and to further develop...

...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors...Our work demonstrates that it is now possible to achieve 20+ fragment assemblies with both robust efficiency...Additional confirmation of accurate assemblies was achieved by the sequencing of plasmids isolated from blue...illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can be manipulated to result...fragment lacI/lacZ cassette assembly was easily achievable with high levels of transformants and low backgrounds...researchers continue to test the limits of DNA assembly approaches with increasingly complex experimental conditions...

...Snapp’s blog post shows. To avoid these issues, Subach et al. and Tsuboi et al. developed monomeric FP...mK-GO from Tsuboi et al. and the FT series from Subach et al.) were derived from previously developed ... while attempting to enhance mKO in other ways, Subach et al made a concerted effort to develop their ...see Table 1 for emission and absorption spectra). Subach et al’s directed mutagenesis also produced the ...Monomeric FP Timers and Associated Plasmids from Subach et al. Protein Excitation (nm) Emission (nm)...following its subcellular localization overtime (Subach et al.) Monitor protein expression dynamics in ... (2001): 29621-29624. PubMed PMID: 11408473. 4. Subach, Fedor V., et al. "Monomeric fluorescent timers...

...application cycle becomes much more complex. “How do I reach out to potential mentors?” “Where do I start with...research that aligns with your interests and goals.  Reaching Out to Potential PIs Even if you’re applying to...application process. Many institutions recommend that you reach out to potential advisors before submitting your...your application that you intend to work with, so reach out to three in case one isn’t able to respond to...your email. During my PhD application cycle, I reached out to around twelve professors and heard back ...you determine if the program is the right fit. Reaching out to PIs also means doing plenty of mini-interviews...applications can get to know faculty, students, and each other. Before COVID-19, financial and logistical...

... that many researchers were having difficulty achieving indel formation (gene disruption) with this new...used by respondents (transfection method, amount of each component per transfection etc.). Take Pooran's ...most of the respondents have been struggling to achieve genome editing with NgAgo, this does not mean the...successful indel generation and epitope knockin was achieved by a few respondents suggesting that NgAgo can...most of the researchers surveyed here could not achieve indels with the NgAgo system indicates that it ...which might be rapidly degraded by the cellular machinery, leaving a small amount of active NgAgo/ssDNA ... cell. As proposed in the original article, to achieve greater efficacy, researchers will have to carry...

...resistance is upon us, and the world is unprepared. Each year in the United States, two million people will...to combat antibiotic resistance using multiple approaches. Major restaurant chains including Chipotle and...bacteria have poor non-homologous end joining machinery, so a CRISPR-induced double-stranded break (DSB...is efficient delivery, as the antimicrobial must reach essentially every cell or else small pools of antibiotic...into the bacterial genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) ...with the caveat that phages are very diverse, and each phage has a narrow bacterial host range. To further...Ando et al. recently engineered synthetic phages, each based on the well-studied T7 phage, to target various...

...incorporate A bases across from abasic sites. This approach resulted in a maximum A → T product purity of ...AYBEv3!). Although AYBE, AXBE, and ACBE do not approach the high product purity of CBEs and ABEs, they...). Several different teams succeeded with this approach around the same time (He et al., 2024; Tong et...al., 2024; Ye et al., 2024; Yi et al., 2024). In each case, a UNG enzyme was modified to recognize T and...editing window than existing CGBEs. This distinct approach means researchers can now select the best tool...although the efficiency, purity, and indel rate of each tool vary considerably. And there’s so much more...on in the base editing field! From CRISPR-free approaches to mitochondrial base editing to base editors...

...easy to ship as plasmids, they still have a global reach. From 2016–2023, we shared 93,491 viral vector preps...Cumulative AAV preps available in the Addgene repository each year from 2016–2023. B) AAV preps distributed per...viral vectors. This process builds on our former approach and involves reading primary literature, soliciting... viral vectors service puzzle: the usefulness of each viral prep in practice. The number of requests shows...redesigned Data Hub in 2022. It’s influenced the way we approach our viral vector educational resources, from eBooks...Cepko, C., Guerin, K. I., Rego, M. A., Ersing, I., Bachle, S. M., Kamens, J., & Fan, M. (2019). Adeno-Associated... Rachel Leeson...

...specific UV wavelengths or chemicals (Polstein and Gersbach, 2015; Zetsche et al., 2015). These types of CRISPR...native conditions, the less activation you can achieve using a CRISPRa. Your gene of interest might already...your system is working as expected. Finally, for each gene you want to activate, you should also be ready...Dow D, Buser-Doepner C, Bertotti A, Trusolino L, Stronach EA, Saez-Rodriguez J, Yusa K, Garnett MJ (2019...engineering parameters via a library-on-library approach. Nat Methods 12:823–826 . https://doi.org/10.1038...JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient...doi.org/10.1016/j.cels.2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system...

...colors to label an organ or entire organism. Because each cell can have multiple copies of the transgene that... colors when a combination of XFP are expressed. Each cell remains the same color for its entire lifetime... as many as 16 copies of the injected transgene, each of which could recombine to activate the expression...expression of a different fluorescent protein. Therefore, each cell could express a unique combination of all of...construct, dBrainbow includes small epitopes fused to each XFP. Secondary antibodies that target these epitopes...onto a tamoxifen-inducible ubiquitin background, each cell in the mouse that expresses Cre undergoes exactly... exactly one recombination event, leading to each copy of the transgene expressing either YFP or CFP, ...