...individual molecules. Poor photon budgets are a widespread issue with fluorescent proteins—even the thriftiest... “ligand” that binds to the tag. Like most good ideas in biological imaging, the initial breakthrough ...breakthrough for in-cell labeling techniques was provided by Roger Tsien, who showed that the selective interaction...short genetically encoded tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure...tags (e.g., SNAP-tag, HaloTag, TMP-tag) – These widely used systems consist of a genetically encoded enzyme...covalent attachment of a fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–tetrazine...fluorophores were designed as antibody and oligonucleotide labels for fixed-cell imaging, not for live-cell...

...the cell might consider junk protein. Though there are a number of chemical and peptide-based proteosome... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...these challenges, researchers have amassed a considerable molecular tool box of tags and fusion proteins...proteins, such as Thioredoxin A (TrxA) that improve disulfide bond formation in order to help keep your protein...bacterially derived in-vivo biotinylation signaling peptide (Bio), flanked by hexahistidine motifs (6xHis) ... a combo tag comprised of a calmodulin binding peptide (CBP), a TEV cleavage site (more on that in a moment...The efficiency of this cleavage depends on the identity of X. A number of other proteases are available...

...and dampness of any kind should be avoided. Distilled water provided to maintain the humidity should be...contamination (Uphoff, 2002). Mycoplasmas are considered to be one of the simplest and smallest bacteria...Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard’ for detecting mycoplasmas...steps start from you. If you are careful enough to provide a sterile environment for work, it will soon become... coughing in the lab. The human mouth contains a wide variety of mycoplasmas and these can enter the cell...you keep all the required equipment and reagents inside the laminar airflow before and while you are working...regularly as mycoplasma, bacteria, and fungus can reside in these containers. Clean up any media or culture...

...so good with video calls, how about trying your hand at a video presentation? Most video platforms make...months past a worldwide shift to work-from-home and just now realizing that we can’t bide our time and ...are moving forward. Here are a few out-of-the-box ideas that might jump start your learning. 1. Create a...and most of us could use more mentors to listen, guide and support us during training and throughout our...see Form Your Own Peer Mentoring Group: A How-To Guide for Scientists.   Once in-person, peer-mentoring...others and get their input as well. Giving a great video presentation is going to be a useful skill for the...learned to play the ukelele...perhaps you get the idea. What will you start? 4. Do informational interviews...

... It can be applied for identification of essential MYC binding sites in a wide range of human cells.  ...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...disease-causing point mutations. They allow single-nucleotide modifications to edit DNA without requiring a...recently deposited a set of plasmids that helps identify base edited cell populations without end-point...reporter for editing enrichment”, or XMAS-TREE, to identify base edited cell populations without end-point...should have changed from “TGA” to “TGG.”  After identification of ABE activity, XMAS-TREE can also be used...difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells are...

...properly onboarded. Most work environments outside of academia provide a formal training regime and the right...transition. In this installment of the Addgene career guide, we’ll think about what causes this culture shock...enjoyable aspect of their chosen career. You can confidently call yourself a Scientist. You work in a lab....during the lab → office transition? I’ve already provided a few counterpoints to some of the causes of culture...colleagues, we’ve come up with a list of things to consider as you make this transition. Of course, a “desk...these--get started with our Transferable Skills Guide.     Over the last decade, my peers and I have... the first place. Finally, if you still find the idea of not holding a pipette again upsetting, there ...

...trying to decide which CRISPR tools are best for your experiment, check out our CRISPR guide or leave ...can be used to modify any desired genomic target provided that (1) the sequence is unique compared to the...Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9...For more details on the PAM site, check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel...series of positive selection screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to ...Kleinstiver et al. 2015). From these screens, they identified four novel SpCas9 variants with altered PAM binding...Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome Biol 19: . https...

...experimental work, but with this guide, we hope you feel more confident incorporating them into your research... We strongly recommend following the protocols provided by the depositing lab whenever available. Addgene...library sample, and any other information or scripts provided by the depositing lab for data analysis.  I'm ... If not, our protocol on Lentivirus Production provides a general approach for producing lentivirus in... the virus–the right amount of virus, that is. Ideally, each cell will be transduced by no more than one...from Addgene? Take a page from our book and put aside an aliquot for titering and optimizing before you... number of cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing...

...Salegio et al., 2013). Zingg et al. 2017 also provide evidence that AAV1 and AAV9 are capable of synaptic...particle is taken up on one side of the cell and released again at another side of the cell after intracellular...Conclusion An ideal comprehensive toolset for neuronal tracing and network identification requires both...single synaptic step, thus preventing further widespread expansion of RABV. This improvement resulted ...delivering neuronal tracers AAV vectors have been widely used by neuroscientists for some time due to their...proteins. For a brief overview see Addgene’s AAV guide. AAV particles are relatively small (20 nm) in size... transgene expression. Fluorescent proteins are widely used for tracing and their expression is often ...

...as a newbie reprogrammer, to decide which method to use. This post provides a brief overview of reprogramming... them with peptides that help mediate their transduction, such as the polyarginine peptide described in...variation between iPSC clones. Retroviruses are not considered safe for clinical applications.     Lentiviral...Like retroviruses, lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox... site of excision, this approach is still not considered safe for therapeutic applications. piggyBac (...insertional mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors ... includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm...

...through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman et al...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...popular tools for genetic screens, enabling genome-wide interrogation of the genes required for cellular...processes. For a successful screen, it is necessary to identify the gRNAs that elicit the phenotype, and this ... the gRNAs responsible for a phenotype can be identified by next-generation sequencing of the genomic ...contributed by Leah Schwiesow A leading cause of death worldwide, cardiovascular disease is driven by atherogenic...ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents (what is commonly called...

...excited to provide ready-to-use CRISPR lentiviral preps to researchers across the globe. Why provide CRISPR...for genes involved in a wide variety of cellular processes. While these are widely requested and incredibly...example, we’ve teamed up with the Broad Institute to provide lentiviruses for delivery of SpCas9 and Cas9 nickase...BRAF and MAP3K4 gRNAs allow you to target these widely studied signalling pathways. Find Validated gRNAs...alleviate some of this labor for our users, we now provide the Brunello (targeting human genes) and Brie (...gRNAs to target around 20,000 individual genes. We provide enough lentivirus for you to both perform an optimization-scale...lentivirus stocks. Our research scientists have provided their standard lentivirus production protocol ...

...scatters sideways to the path that it was initially traveling is called side scatter (SSC) and provides information...their observations. However, while flow cytometry provides analysis at the cellular level, it cannot be used...degree of scatter is captured by detectors and provides useful information about the cell. Light that ... traveling is called forward scatter (FSC) and provides information about the relative size of the cell...reporter on the N- or C-terminus of the protein. One downside of fusion proteins is that the tag can alter the...granulocytes in a plot of forward scatter (FSC) versus side scatter (SSC). Users can gate around their desired... analysis, include a complete set of controls. Ideally, every flow cytometry experiment will have a negative...

...Madison lab created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA...well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using...fluorescent reporter. Lastly, once a mutant is identified, the PB transposase can easily and seamlessly...fluorescent protein for assessing CRISPR/Cas9 activity Guide RNA structure and the site in the genome you are...Sander and Joung, 2014). Thus it is essential to identify the best sgRNA to ensure success in your CRISPR...-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired ...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of mCherry...

...appropriate for your specific research question. Considerations when choosing your Cas nuclease When choosing...experiments, there are three important factors to consider: site accessibility, specificity, and sensitivity...cleaves a site that is not a perfect match to the guide RNA (gRNA) used to target the genomic locus of interest...interest. As these off-target effects can be genome-wide and, in some cases, difficult to determine, the ... off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity...measured by calculating the on-target activity divided by the off-target activity. Figure 2: Specificity...engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al., 2018). The David Liu...

...himself from the idea of starting a company around engineered bacteriophages. He decided to quit his "safety...Operations of Sample6, for advice on how to take an idea developed during graduate school and turn it into...into a biotech company. Transitioning an idea from academia to a startup Mike Koeris and fellow co-founders...co-founders Tim Lu and others first came up with the idea that would evolve into Sample6 as graduate students...using bacteriophages as bacterial therapeutics. This idea was incorporated into Novophage Therapeutics, Inc...that entrepreneurs must clearly communicate their idea at various levels of complexity and abstraction ...people with diverse backgrounds how amazing their idea is. While communication styles in academia and industry...

...size, the cytometer provides you with two measurements: forward scatter (FSC) and side scatter (SSC). To... being considered for the analysis. (C) A more liberal gate allows more cells to be considered for the... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage...scattered forward at a low angle (FSC) or to the side at a large angle (SSC). FSC and SSC depend on the...three gates will likely be: the FSC/SSC gate to identify cells, the single cell gate to exclude duplicate...running my flow analysis, I can however clearly identify my lymphocyte population (10.7% of all recorded...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...

...structure of the POI would work best. Ideally, structural information can guide this decision. In our experience... This is an important downside of the technology and should always be considered when interpreting results...that is strong evidence that the fusion behaves well. But this is not conclusive evidence that the fusion... any tendency of GFP to homodimerize should be avoided because it will double the size of the protein....or (ii) designed to form an inert, unstructured peptide and therefore consisted of glycines, serines and...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...al., 2011).  Although structural information may guide design, it is advisable to generate multiple constructs...

...clinical applications could be considered. It is important to consider the question of broader applicability...underwent repair not from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but...allele rather than the supplied single-stranded oligonucleotide (ssODN) template. Embryos also underwent editing...failure of the embryos to undergo editing using the provided ssODN template. In fact, this finding led the ...conducting research in embryos should our society decide that germline editing is permissible in cases of...disease prevention. However, it is important to consider that already-tested CRISPR alternatives are applicable...target. In addition, as on-target editing rates vary widely from gRNA to gRNA, it’s unclear where this study...

...week. Here we will provide an inside look at the steps we take to verify the identity and quality of the...plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through...the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids... ends to verify that the sequence assembly and identity of the insert are correct. In other cases, we ...glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then select forward...corresponding glycerol plate on dry ice to the sequencing provider. Each sequencing service will have different requirements...requirements for volume and concentration, and not all providers are equipped to perform Sanger sequencing from...

...Editing Guide RNAs Download the CRISPR 101 eBook Resources on Addgene.org Read the CRISPR guide Find CRISPR...tools. These papers are chock full of great tips to guide your experimental design, especially the design ...nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA consists of a CRISPR gRNA...successful, although sequences with G/C content outside this range can still be optimized (Anzalone et ...transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even more important...influence the success of your prime edit. If possible, consider following these suggestions: Edit the PAM along...DNA mismatch repair (MMR) is less efficient at identifying these “bubbles” of mismatched bases, enhancing...

...to start considering my future path. Talking to Addgenies has helped me get a much better idea of what ...organization beyond my job description.” Our resident bagel provider, source of energy, and conversation master...friends in the Biochemistry major seem to have the idea that if you don’t get your PhD you’re set up for...undergraduate school and studying for the MCAT, she decided that she didn’t want to take it. “I realized if...to her. Addgenie Shreya Vedantam with our resident dinosaur! Read her Instagram feature here. ...scooped, she said. Reflecting on her values, she decided that this “is not [her] view of science. It should...joined an investment club and discovered the business side of science, she stuck with it. Lab Manager Shannon...

...get an answer, consider asking to avoid unrealistic expectations. And finally, provide feedback. If ... in customer service, I've daydreamed about the ideal customer service interaction - one that gives our...experience is based solely on my time at Addgene, I’m confident that these tips and tricks will apply to any biology-related...Your time is precious. Here are a few things to consider before you commit: Double check to make sure ... it’s not in the best interest of any company to hide information about their products from you, unless...papers that successfully used said plasmids. We provide these references as our way of saying “Hey look...can be a particularly good resource if you are considering using a reagent for “off-label” purposes. When...

...individual gRNA from this single vector) and guide-only (lentiGuide expressing only an gRNA, for use with cells...new model or a new assay or both, and you have no idea what genes are involved. Or you don’t know how well...Or you don’t have enough cells to execute genome-wide perturbations. So, while some might argue that the...the CRISPR kinome library? Thus, we are glad to provide CRISPR libraries targeting the mouse and human ... a well-annotated collection of genes that can provide a useful starting point for developing a model ... libraries are available from Addgene as pools provided in two sets, each with 4 gRNAs per kinase, so ...opposed to the 70,000 or more gRNAs found in genome-wide libraries, and thus can be screened in pooled format...

...’s possible to get an idea about the subcellular structures your protein resides in and potentially which...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...transport between healthy and diseased states can also provide interesting insights into disease mechanisms and...in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth review of quantitative colocalization... and well-executed experiments require some consideration of FP properties. It is important to look out...Barbier Lab can be used to fluorescently label a wide variety of gram negative bacteria (West Virginia...Virginia University School of Medicine, 7). Identifying the subcellular localization and composition of cellular...

...with people inside and outside your field. Your colleagues, lab mates, and PI can provide feedback on ...poster – now it’s time to practice your delivery. Consider your audience: Who will be attending the session...audience so that they can digest what you’re saying, provide constructive criticism, and ask thought-provoking... for Cancer Research Annual Meeting, you might consider including more background on how your virus of... points will help you remember what to say and provide your audience with a clear picture of your work...on your technical presentation, but those outside of your field can point out places where you gloss over...you’re standing at the session. Present with confidence Now that you’ve printed your poster and practiced...

...though it has been shown to identify trends correctly (Muhamad, 2011;  Providenti, 2006). Its biggest advantage..., look for the lowest-cost option that provides you confidence in the results you need.  Whichever ...Taqman’s increased sensitivity allows for a more confident determination of copy number, albeit within a ...to a standard curve, and most results can be confidently interpreted as integers. Since each sample is...approach is right for your experiment. You’ll have to consider:  Resources needed Most molecular biology labs...exact copy numbers or just general trends? How confident do you need to be in your determination to move...determine exact copy number and it may not be able to identify trends in cases where the copy number variation...

...out Addgene's western blot protocol and protocol video!  Antibodies The most important part of your western...  Lysis buffer When choosing your lysis buffer, consider the subcellular location of your protein. Proteins...SDS-containing lysis buffer, like RIPA, should be considered if your protein is difficult to solubilize, such... protein of interest is small, you may want to consider a smaller pore size (0.2 µm). Conversely, if your...well for proteins of most sizes and are worth considering if your proteins of interest vary wildly in size...used for multiplexing, and the solutions used to provide the chemilumescence may be light-sensitive. They...size. However, HRP is not compatible with sodium azide, a common microbial agent used in antibody buffers...

... CRISPRi screens to identify essential genes and phage host factors, and identify sequence-specific toxicity... inducible CRISPR/Cas9 activation E. coli genome-wide inhibition library CRISPR knockout libraries for...break at the genomic loci of interest and on either side of the cargo (ex: fluorescent protein gene) on the... donor plasmid cargo are complementary to either side of the double-stranded break in the genomic loci...the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the last edition of “What’...in CRISPR,” we shared with you an E. coli genome-wide inhibition library from the Chong Zhong lab. Now...Now, we’ve included another E. coli genome-wide inhibition library from David Bikard’s lab. The Bikard ...

...Protein-based tags typically provide better labeling specificity than short peptide tags like tetracysteine...forgot it was already in the freezer… you get the idea. What if there were a way to just make one fusion...it takes is a bit of chemistry magic to produce a wide range of useful molecules linked to that substrate... the photophysical properties of chemical dyes provide many advantages over fluorescent proteins, opening...1998 (Griffin et al., 1998), this 15 amino acid peptide contains a cluster of four cysteines (CCXXCC, where...using engineered ligases to attach a ligand to a peptide tag on your protein of interest or using non-natural... protein is functional. In some cases, a small peptide tag like tetracysteine might be the only option...

...transposons due to changes in coloration of corn kernels. Aside from corn, transposons are in the genomes of many...encode a transposase enzyme.  Class I TEs are considered replicative since every time they jump they make...transposons are used in the lab, the transposase gene is provided in trans so that a gene of interest can be inserted...preferred target site for integration is TA dinucleotides and it leaves behind the CAG DNA footprint from...with next generation sequencing, and analyzed to identify which insertions were positive or negative selected...site preference and host range are important to consider when selecting a transposon for a mutagenesis ...regions, gene disruption is less of a concern. RNA guided transposons insertion One drawback to DNA transposons...

...reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective...In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively easy to transduce...news for a screen. Because Addgene viruses are provided at a specific titer, you can better plan your ...more difficult to get them to grow out. The only downside is that it takes a few weeks for single cells ...Using this virus, it’s expected that Cas9 activity, guided by the virally-delivered GFP gRNA, will edit out...products (which could be easier to visualize and identify). If you need/want to put the digestion site in...of the PCR product, you would still be able to identify the cleavage product. 3. The Surveyor kit had ...

...quarantining {% video_player "embed_player" overrideable=False, type='scriptV4', hide_playlist=True, viral_sharing...an estimated total of 380,000 deaths worldwide (statistics provided by Google on 6/4/20). Many countries...interested in examining the effect of COVID-19 on worldwide scientific activity. However, this type of open-ended... requests over 6 years for 35 papers (data was provided by Addgene on 5/26/20 and contains requests from...holiday effects. Based on these observations, we considered the following (noiseless) model for our data:...factor 𝜙z if t is during event z. The "events" we considered for our model were weekends, Christmas/New Years...Europe. To improve the stability of our model, we considered only the 10 papers with more than 10 unique order...

...meeting and a means to request funding for events, consider reaching out to it. PTAs have more flexibility...Science Night. There are plenty of places to get ideas for science activities. I did some crowdsourcing...teachers socially and we’d already discussed the idea of my giving a CRISPR presentation to their students...students learned something. Based on the initial guidelines I received from the teachers, I formulated the...schooler himself and b) I had the background to be considered an authority in this area. What is genome engineering...CRISPR work? This section was essentially background slides getting everyone on the same page in terms of the... by running it by the teachers and sharing some slides on Twitter, where I was able to get some great ...

...watch a how-to video. My dad, on the other hand, mastered the art of finding how-to videos on YouTube long...today's science communications: podcasts, blogging, videos, lectures, exhibits, social media… how do you choose...of looking for The One Method to Rule Them All, consider the following four questions to figure out which...use at Addgene: YouTube, for longer educational videos, and Twitter, for sharing updates, new deposits...precious free time.  And don’t be afraid to think outside of the box. There are so many ways to communicate...comic about math probably didn't sound like a great idea to most people in 2005, but XKCD is pretty successful... can make for beautifully compelling photos and videos, while molecular, micro, and cell biology are often...

...many different inserts. The downside: this type of primer does not provide information about the orientation... blog post discusses some of the key things to consider when performing colony PCR.   Designing colony...different inserts. When you design primers to anneal outside the cloning site, it doesn’t matter what the sequence...information about insert orientation, then you might consider designing orientation-specific primers. Blunt ...primer pair for each insert Backbone-Specific Provides information about the size of the insert and if...because primers are plasmid dependent Doesn't provide information about the orientation of the insert...-up Setting up colony PCR reactions is nearly identical to preparing a standard PCR reaction: combine ...

...organization or a program and a funding opportunity coincided, and they stepped up to apply for a grant which...to evaluate the organization’s grant readiness, identify grant opportunities, secure grant awards, and ...receive and manage funds.  Grant professionals identify grant opportunities with the highest likelihood...strategy and projections around that likelihood. We provide expertise on the processes needed to successfully...grantmakers. We keep up-to-date on the changing guidelines and regulations impacting grant management, we...career path changed when I was asked if I would consider consulting for a particular grant by another state...transferable. Use your experience and your passion(s) to guide you in building a grant career. Do you have a passion...

...the use of chemical pesticides may be avoided or decreased. Biosensors for herbicide detection Dr. Rodrigo...scientists work on projects spanning from the identification of plant compounds and insecticidal proteins...renewable biotechnology products and biosensors for herbicides as well as developing yeast as source for dietary...focuses on endophytes, bacteria, and fungi that reside within living plants and that produce compounds...expression system pDawn to clone and express newly identified proteins and enzymes. These compounds will later... yeast capable of desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc...thrives to make affordable biosensors that detect herbicides, such as glyphosate, in water. He aims to leverage...

...said to be hyperpolarized. The chloride pump NpHR brings in negative chloride ions which hyperpolarize the...generation times and high fecundity, make zebrafish ideal for high throughput studies of optogenetics.  ...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...inhibition: Hyperpolarization Conversely, when the inside of the neuron becomes more negative it becomes ...swimming. Zebrafish behavior studies in action: Identifying neurons that trigger spontaneous swimming  Researchers...lab applied optogenetics in zebrafish larvae to identify new neurons that trigger the CPG (central pattern...

...it is crucial for scientists to not only easily identify base editing events in real-time but also enrich.... APOBEC-Cas9n-UGI targets its preferred trinucleotide motifs (5’-TCA-to-TUA) found within the 43 base-pair...cells expressing eGFP and mCherry. ACE was used to identify cells that have been successfully edited with ...efficiency in real-time, it relies on DSBs which is not ideal for monitoring single base edits (St. Martin et ... cells. mCherry is constitutively expressed to identify successfully transfected cells. C-to-T editing...2019). TREE is a real-time, fluorescent based identification system for the isolation of base-edited cell... plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and subsequent...

... past decades, neuroscientists have made great strides to map brain regions and allocate specific functions... modulating. Viral vectors, in contrast, are considerably more versatile and specific in how they manipulate...manipulate neuronal function, with far fewer side effects compared to lesioning methods -  they may be the...ve previously discussed some of the general considerations for performing behavioral tests, but now we...used since the 1970s to test the efficacy of antidepressant treatment in rodents. In this test, an animal...BDNF in the hippocampus causes a shift towards antidepressant behavior in rats in the forced swim test as...neurons (Drd1) in mPFC is responsible for the antidepressant effect. To specifically activate the Drd1 neurons...

...transformed E. coli expressing Cas7-11, they found no evidence of growth inhibition when the system was retargeted... more they worked with it, the more they found evidence contradicting their initial assumption. Eugene... Type III-E, and then used this information to identify a large variety of related bacteria with similar...with lower knockdown efficiencies. The ability to identify related Cas systems opens up the potential of ...proteins and a putative Cas11-like protein, and a guide RNA. The enzyme has dual activities: processing ...CRISPR-driven precision into a genome, can be considered the fourth generation of CRISPR, one which drops...drops itself neatly into that gap. Consider, for instance, a disease like cystic fibrosis, which can be...

..., with residence times on DNA of approximately 6 hours. To our surprise, we measured identical dissociation...With 36bp on the PAM-distal side of the break and 91bp on the PAM-proximal side, consistently supported ... Jacob Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The...DNA post-cut. Using substrate DNA labeled on both sides of the break, we determined that Cas9 held tightly...introduces functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from ...PMC4383097. 3. Kim, Sojung, et al. "Highly efficient RNA-guided genome editing in human cells via delivery of purified...HDR Basics Learn How to Design Your gRNA Find the Ideal CRISPR Software for Your Needs Resources on Addgene.org...

...Cycle Quick Guide to All Things Lentivirus More resources on addgene.org Lentiviral Guide   Popular lentiviral...staple in molecular biology for over three decades. Widely used across various research applications, they..., for some applications, integration might be considered a double-edged sword with unintended consequences...detailed explanation, check out Addgene's Lentiviral Guide). To produce lentiviral vectors, the lentiviral ...AKA transduction in the viral vector world). Once inside the host cell, the viral payload is integrated ...especially concerning in clinical settings, as evidenced by retroviral gene therapy trials for X-linked...nucleic acid for only a limited time, IDLVs are ideal. As the episomes are lost during cell division, ...

...in industry. The news is good. In their ideal form, ELNs provide users with a holistic means to manage ...will always be a part of everyday work to capture ideas, write down certain notes or brainstorm, but we ... able to understand the situation in depth and provide the solution that fits the most labs, our team ...of budget and ease of use of the software. We identified that when talking about ELN costs and limited... to require on-going maintenance and support.” Besides that, academic institutions tend to think about...intellectual property across various software providers used to supplement their paper notebooks. Instead...witnessing and audit trails. How does the ELN provider protect and respect your IP rights and data ownership...

...we’ll go through some of the most important considerations to make when planning and performing an IF ...try the Introduction to Fluorescence Microscopy video from iBiology). IHC and ICC are essentially just...antibodies is a time consuming and expensive process. Consider the vast number of possible primary antibodies...experiment The process to perform IF can be broadly divided into the several steps outlined below and in Figure...steps include: counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview...indirect immunofluorescence procedure with key considerations about each step. Image created with BioRender.com...’ll start there. Some of the most important considerations are to make sure your secondary 1) will recognize...

...is generally error-free provided the DNA template used for repair is identical to the original DNA sequence...directed repair pathways HDR mechanisms can be divided into two main camps: non-conservative and conservative... many applications. Your favorite Cas enzyme, a guide RNA to your site of interest, and a donor DNA molecule...you need to generate a knock-in.   General considerations for designing a repair template to create mutations... modification should be very close to the DSB, ideally less than 10 bp away if possible. Check out our...ssDNA templates (referred to as ssODNs (oligodeoxynucleotides)) are commonly used for smaller modifications...cells than their plasmid counterparts. Another consideration for dsDNA templates is their ability to randomly...

...information, see the manual provided by the Weiderschain lab. Find Tet-pLKO-puro. lentiGuide-Puro - This plasmid...infect many different cell types so it is useful in a wide range of applications. This plasmid was the top ...sgRNA scaffold and cloning sites for inserting the guide sequence (pSpCas9(BB)). The 2A-GFP allows for screening...produces a ~10 fold higher titer virus, making it ideal for CRISPR pooled library screening experiments....Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome engineering. For use in human...annealed oligos can be cloned to create a chimeric guide RNA. Find pX330-U6-Chimeric_BB-CBh-hSpCas9. pSpCas9...with cell lines already expressing Cas9. Find lentiGuide-Puro. pSpCas9n(BB)-2A-GFP (PX461) - Similar to...

... encoding multiple guides. While two guides require two arrays for Cas9, two guides can be streamlined...enAsCas12a), minimizing off-target effects by identifying guide selection rules, and reducing recombination...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...optimized genetic screening technique, researchers identified a synthetic lethal interaction between MARCH5...Cas12a-based screening method was used to create a genome-wide library capable of performing similarly to Cas9 ...target multiple genes within a single cassette provides increased simplicity for combinatorial genetic...cost-effective alternative to commercial ladders which also provides a useful yet low-pressure teaching opportunity...

...of this pathway and does not consider the missing or damaged nucleotides that are common to biological... in Mammalian Cell Lines [Video] Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional...less heterogeneous when constrained by sequence identities in flanking sequence (“microhomologies”). NHEJ...the Cas9 DSB (blunt or near-blunt ends without nucleotide damage) such products are rare, probably accounting...of the canonical Cas9 approach. A pair of CRISPR guides that flank regions of hundreds of base pairs or... by NHEJ-dependent repair (“pop-in” insertion) provided a template containing compatible overhangs is ..., Zhang Y, Zhang F (2013) Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity...