...downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case, the ...there are 10 putative off-target locations you can 1) determine if editing at those sites would confound...While this may effectively serve as a transfection or transduction control, a different gRNA would have..., you’ll want to select a Cas enzyme with low tolerance for mismatches within the gRNA sequence. ...near each other, this will result in a DNA double strand break (DSB), the event required for frameshift ...

...in the crystal jellyfish Aequorea victoria (Figure 1) in molecular detail. On the underside of the “umbrella...glowing protein could be used to measure gene transcription, as well as to track protein localization. And...bioluminescent, some types of corals and anemones are vibrantly colored. He hypothesized that GFP-like proteins...

... science 1)      In February, the Office of Science and Technology Policy issued a memorandum calling ...(66 percent) gave preference to journal access granted via a public repository or authors’ own web sites...

... and a reverse transcriptase (RT) template including the desired edit. Figure 1: Prime Editing ...doi.org/10.1038/s41587-021-01039-7 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...although sequences with G/C content outside this range can still be optimized (Anzalone et al., 2019). .... Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides. For...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...

... same output regardless of who or which computer ran the process. Another design choice we appreciated...drives our bioinformatics needs       Fig. 1: QC'ing data at Addgene is like standing under a Niagara...automating the bioinformatics steps needed to transform the raw sequencing data into a complete plasmid...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect...

...changes.   1. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection by Alyssa...editing. Using a fusion between Cas9 and reverse transcriptase and a prime editing guide RNA, it can introduce...

...other. Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base...SpCas9 base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated...engineering and protein delivery. Nature Communications, 8(1), 15790. https://doi.org/10.1038/ncomms15790 Richter...the opposite strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are ...place on the free strand of DNA and converts a C to U without producing a double strand break. UGI = uracil...repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared...are base transition editors: converting one purine base to the other or one pyrimidine base to the other...

...Consistency, and Transparency. This label scores products by their environmental impact on a scale of 1-10: the... but with the right incentives (for example, by grant agencies), we can shift toward a more sustainable...your RMV to the landfill, it’s collected and transferred to the Red2Green processing facility, where it...

...organized and boost your productivity in 2014. Tip 1: Use color coding, labels, and post-its to make finding...pile of lab notebooks and binder on your desk. Arrange them in chronological order (or by project, if ...Organize you experiments, keep track of upcoming grant deadlines, and sync everything to your calendar....lists of your upcoming tasks, set deadlines, and arrange automatic email reminders. These programs can be...

... western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using ...using antibodies.   Figure 1: Overview of the Western blot process. The sample, a mixture of protein...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally visualized...a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated...migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely submerged...filled with a transfer buffer formulated to conduct the electric current. The wet transfer process is very...

...also available for lentiviral transduction. Lysine-specific Demethylase 1 (LSD1) Tatjana Sauka-Spengler's...P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter...light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases...CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic...other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš... PuroR enable sorting and selection of transduced cells. Grant Challen’s lab also created constitutive...associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret...

...Presented in figure 1). More information on Agrobacterium-mediated transformation can be found in this...interest. Figure 1: Simplified schematic of agrobacterium-mediated transformation in plant cells. Agrobacterium...variants (hereafter referred to as Cas9) and a single stranded guide RNA (gRNA) is sufficient to modify the genome...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...CRISPR components can be expressed stably or transiently depending on the delivery method and cell type...CRISPR components can be delivered and expressed transiently using a standard detergent, Polyethylene Glycol...plant genome. B) The Ti plasmid and helper are transformed into agrobacterium tumefaciens and exposed to...

...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse of...cellular analysis products at Promega.     References 1. Hall, M.P., et al. (2012) Engineered luciferase reporter...process known as bioluminescent resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating... consists of NLuc fused to the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent...for excitation. The result was the cyan-excitable orange fluorescent protein, CyOFP1. CyOFP1 was molecularly...and improved maturation time compared to similar orange fluorescent proteins. In their work, the authors...great imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide ...

...relations, advocacy, and writing legislation (see Figure 1 for career map). The beautiful part of science policy...about science policy as a profession. Figure 1: Science policy can lead you to many paths! For a ...AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even...Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I could never get one straight...variety of backgrounds with technical expertise that ranges from astronomy to bioethics to chemical engineering...happen at the national level within the executive branch or in Congress, or can also happen at the state... early career researchers who have decided to transition out of academia from graduate school or a postdoc...

...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...sharing even broader and began working with tech transfer offices to make reagents available to scientists...

...communication check out these SciComm blog posts. References 1. Bourdeau, Raymond W., et al. "Acoustic reporter genes...topics both interesting and approachable!   The grand finale of visual impressions was delivered by the...

...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system: a vector...contains the MISTIC (membrane-integrating sequence for translation of integral-membrane protein constructs...glycosylate proteins and may recapitulate the post-translational modifications added to proteins in other eukaryotes...includes a special feature for difficult-to-purify membrane proteins. pCri vector pCri-13a, designed for use...protein tag. MISTIC drags tagged proteins into the membrane and may improve expression of proteins that natively...

...Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle M., et al...ve also carved pumpkins (yes, Blugene made an appearance there too) and shared fascinatingly creepy science...algae cell is just the cell wall and cytoplasmic membrane. Vampirovibrio chlorellavorus will not grown in... the snail, the larvae reach the eyestalk and transform them into a pulsating display imitating a moving...grasshoppers and crickets. When it’s ready to transform into an aquatic adult, it forces its insect host...uninfected grasshoppers, including those linked to neurotransmitter activity and geotactic behavior (Biron et ...

...proteins, DNA, and RNA used in CRISPR.   Figure 1: Overview of the parts of CRISPR. The bacterial chromosome...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence... and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one...protospacer-adjacent motif” present in the non-target strand of DNA to be cut, which varies between CRISPR systems...from Addgene!   RNA crRNA: “CRISPR RNAs” are transcribed from the CRISPR array and include a spacer and...

...of experiments they are commonly used for.  Table 1. Points to Keep in Mind When Performing Biosensor ...enzyme or transcriptional activation, conformational changes, translation, and protein translocation. Targeting...triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins. FRET is highly...characteristics like pH, redox state, and voltage across membranes. These sensors generally follow the same pattern...an organelle-specific targeting signal or a transmembrane domain, biosensors can be targeted to a particular...endoplasmic reticulum (ER), vacuole or cellular membranes. Examples include blue flame mitochondria-targeted...

...strategies in the comments section!   References 1. Yearly Planning Meetings: Individualized Development...then formulate a plan to further develop your transferable skills and reach your career goals.      Although... What Makes a Good Manager Anyway Developing Transferable Skills During Science Training ...

...fingers of a completely normal size.      References: 1. Doench, John G., et al. "Optimized sgRNA design to...end with little more than an Excel spreadsheet of random numbers that’ll leave you, well, #sad. Learn...

...et al., 2019; McLaughlin, 2019).    Figure 1: Schematic of multiplex immunofluorescence approaches...). Springer New York. https://doi.org/10.1007/978-1-4939-8935-5_26 Jambor, H., Antonietti, A., Alicea,...palette for display (like green-magenta-blue or orange-blue rather than red-green). Even better, show ....2846 Radtke, A. J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R...

...Kan, SpHIS5, or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent tags in S. cerevisiae...life repair genomic damage, specifically double strand breaks. Researchers have long taken advantage of...product is introduced into the cell by direct transformation. A given insert will typically contain both...selectable gene product for isolation of successful transformants. Addgene distributes several ready-to-use, modular...

...that work for our experiments!)     Figure 1: The IBEX Imaging Community front page. That’s...data, more discussion, and more functionality. Transparency, both in what works and in what doesn’t, and...Ichise, H., Gola, A., Kabat, J., Lowekamp, B., Speranza, E., Croteau, J., Thakur, N., Jonigk, D., Davis...00644-9 Radtke, A. J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R...

...plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in Caulobacter cells ...The system consists of dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool that...CRISPRi plasmids A multiplexed gRNA piggyBac transposon system for human pluripotent stem cells Lindy...and gRNAs whose expression varies depending on transgene design and delivery. They generated hPSC lines...

...save that application for another day! References 1. Tanenbaum, Marvin E., et al. “A protein-tagging system...better. More transcription factor binding sites in a promoter lead to higher transcriptional activation....for single molecule imaging, finding that plasma membrane-targeted CAAX-SunTag was 18-fold brighter than...

...social media? No problem - see our recap below! Day 1: gRNA design tools: How to Design your gRNA for CRISPR...or A:T->G:C edits in DNA without causing double-strand breaks. Our new page shows you base editing plasmids...certainly taken notice! Our newest page in the Transcriptional Regulation category already has five blue flame...with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation: Truncated gRNAs for Regulating ...

...depends on what you are looking to study. Figure 1: A schematic showing the differences in chemogenetics... have pushed the boundaries in neuroscience by granting targeted control over neuronal activity. While... Mouse studies, however, require a permanent intracranial implant to be surgically installed to allow ...receptors Invasiveness Mice require permanent intracranial implant Noninvasive Controlling Stimulation...

... towards understanding this disease. Figure 1: Scientists are continuing to do essential research...community response initiatives. Accelerate the translational impact of research Universities have also been...creating research synergies and accelerating the translational impact of research activities. By streamlining...

... everyone was very approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition...with a detailed training schedule that helped me transition easily. Training and responsibilities Through... the “9-5” was not as scary as it seemed. The transition into a traditional work environment was aided...

... successful postdoc fellowship. 1. Reverse engineer success On Day 1 of your postdoctoral experience,...postdoctoral fellows (PFs) face a number of challenges ranging from long periods of training to limited job opportunities...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary data to...your own lab and get an independent investigator grant (e.g. an R01 from the NIH, in the United States)...postdoctoral mentor. A good mentor typically writes the grants that fund your research, guides the research directions... available for mentees due to travel, teaching, grant writing, etc. Also, it is unlikely that a mentor...reference, informal recommendations, more positive grant application and manuscript reviews, etc. You'll ...

... antibody recognizes and binds to.   Figure 1: Labeled diagram of an antibody including Fc, Fab,...45. https://doi.org/10.1016/B978-012198382-6/50026-1 Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin ...weight by SDS-PAGE, and are subsequently transferred to a membrane. A primary antibody recognizing the protein...is added, and will bind to that protein on the membrane. Then, addition of a secondary antibody allows...showing GFP expression in cells that have been transfected with a plasmid encoding GFP. Actin, a common ...bind to proteins on the cell surface, or the cell membrane can be permeabilized to allow antibodies to bind...

...you do just this, probably in less than a minute (1). Green Listed is a new software tool used to design...found through https://wermelinglab.com/.   References 1. Panda, Sudeepta Kumar, et al. "Green listed—a CRISPR... modified cells are transferred into an animal and the behavior of the transferred cells is followed. ...generating a massive amount of descriptive transcriptomics data. Custom CRISPR screens designed to target...pathway with many receptors, signaling molecules, transcription factors, and ligands. The presenter then showed...

...updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric G protein dissociation...-visual G protein signal transducers (collectively known as the transducerome) that can be activated downstream...Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which...non-visual G protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing...including smell, taste, sight, inflammation, and neurotransmission. But with approximately 950 different human...involved in sight (Olsen RHJ et al., 2020). A transducer complex consists of a trimer of three subunits...such a tool is being able to identify specific transducer complexes that are activated in response to a...

...the most out of their meetings. Amanda Gunn ran randomized coffee trials for us at ASCB|EMBO 2019, matching... Figure 1: The Binning Singletons logo. Image: James et al., 2020. Help me stranger - Finding mentors...within a bin as Horizontal Transfer. This comes from horizontal gene transfer, which occurs when bacteria...in Atlanta, GA, in 2018. A lot of my experiences rang true with theirs.  It was at this dinner and the...

...enables the high-resolution tracking of more than 1 million cancer cells under drug treatment.” Stegmeier...members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe. We’re excited...members and get to know them! Khandan Baradaran, PhD Khandan Baradaran’s expertise in viral safety, gene therapy... with health authority regulations,” Baradaran says.  Baradaran’s path to science began in high school... getting these reagents is profound.” Frank Stegmeier, PhD Frank Stegmeier is the Chief Scientific Officer...in viral genetics. In regards to open science, Baradaran thinks the best way to solve problems is to work...think will get the research funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several...

...brain where DA quantitation is desired (Figure 1). The probe perfuses liquid, which causes the passive diffusion...dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous role as the “happy...standard method of measuring DA and other neurotransmitters in the brain has been in vivo microdialysis...performed with a probe surrounded by a semipermeable membrane, which is inserted into the region of the brain... diffusion of DA across the semipermeable membrane from the extracellular fluid into the perfused liquid... to a threatening looming stimulus in vivo in transgenic zebrafish Report DA release in live mice over...developments in the detection and measurement of neurotransmitters in various species and while exhibiting complex...

... to just that particular assay.      Fig. 1: A native protein (left) is denatured, allowing the...bound to anything? Is it a transmembrane protein, or is it know to translocate or fold in certain conditions...as part of performing a specific function. A transmembrane protein that is being used as a marker for flow...cell, instead of when it is attached to a cell membrane.  Picking the Right Antibody  It can get even ...looking for a protein of interest while it’s in a transition state or specific configuration. So how do you...

...vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check out our complete guide...). If you can get away with generating transiently transfected cells, you will not need a mammalian selection...mammalian selection cassette to screen for positive transfected cells. The addition of an in-frame fluorescent...create a new strain with a disrupted gene by transforming wild-type yeast with an integrating vector expressing...majority of fly vectors are based on p-element, random or targeted, integration into the fly genome and...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...

...materials to the research community. References 1. Coroadinha, A. S., et al. "Effect of osmotic pressure... AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral vectors ...to be due to the viral particle’s affinity for heparan sulfate proteoglycans on the surface of the packaging...supernatant, the viral particles associate with the cell membrane and are taken into the cell via endocytosis. In...looking to improve their AAV yields. Given the wide range of downstream applications scientists rely on our...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show that there...test the endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe...

...ScFvs, based on community demand. Figure 1: Antibody against PSD-95 from the Trimmer Lab. Information... our values of transparency, reproducibility, and community-building. The transparency begins with sharing...Addgene’s antibody resource is funded by a generous grant from the NIH’s Brain Research Through Advancing ...share plasmids with each other. Together, we can transform the antibody sharing landscape as well.     References... Improving Reproducibility through Rigor and Transparency of Experimental Methods. Neuron 90(3):434-6.... project is supported by a NIH BRAIN Initiative grant (U24NS119916 ). The content of this article is solely...

...the life scientist or bioengineer.   Figure 1: Map of a plasmid with its elements described below... origin of replication (which controls the host range and copy number of the plasmid) and typically include...for further study. Promoter Region Drives transcription of the target gene. Vital component for expression...cell types. Primer Binding Site A short single-stranded DNA sequence used as an initiation point for PCR...vector containing the newly inserted gene is transformed into bacterial cells and selectively grown on...biologists to broadly utilize plasmids across a wide range of applications. Some common plasmid types include...

...plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system. Genome wide mouse...target cells that have previously been stably transfected for Cas9 expression. The Yusa lab has used their...

...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure...preprints, data, software, micropublications) in grant proposals. ReFigures are one such research product...

...could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery box...ahead of time included contacting YNP staff to arrange permits to visit places not accessible to the public...night was fun but also very tiring. It feels a bit strange to be using a micropipette and gloves at a campsite...similar to a type II-C Cas9 from Parvibaculum lavamentivorans, whose optimal growth temperature is 30°C. ...rRNA in bacterial RNA-Seq. This enriches for transcripts of interest without an increase in the number...

...into three main effector functions.  Figure 1: Fc effector functions in an immune response to a ... the pores being opened in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive... survive when holes are punched in their membranes, the pore'd pathogens quickly lyse and die.  Antibody...where the eosinophils release cytotoxins via degranulation.  Antibody-dependent cellular phagocytosis (...

...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ...of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals...for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...cooperate to cause a higher-energy electronic transition in a fluorescent molecule (see figure to the ...past 15 years efforts have been made to create transgenic mice expressing fluorescent protein reporter ...microscopy enabled scientists to see how chaotic and random the scanning was and how fast T cells could jump...Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed...

...targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...sonification. Though it helps capture weaker or more transient DNA-protein interactions, the reliance on fixation...McKay lab we study how differential access to transcriptional regulatory elements is controlled during development...

...in online patient/caregiver communities  Table 1: Examples of defining your audience for science communication...  For example, while “PCR test” has recently transitioned from jargon to non-jargon in the context of ...number of characteristics. Common ones include age range, familiarity with the subject, shared interests,...