..., such as chemotherapy treatment or ionizing radiation, can also result in dsRNA generation. While these... Here, we will cover how these structures are generated, why it’s important for cells to sense their formation...these viruses have one thing in common – they can generate dsRNA as part of their infection and/or replication...inherently double stranded, while ssRNA viruses can generate dsRNA intermediates as part of the viral replication...mRNA. These RNA tools have been used in clinical therapy to target pathogenic proteins, as well as in the...as long/short interspersed nuclear elements can generate dsRNA by bidirectional transcription or as inverted...is synthesized from a DNA template, inherently generating RNA:DNA intermediates in the process. These transcription-coupled...

...complementary site in the genome where it will proceed to generate a blunt DNA double strand break. Cas9 has been...Cas12’s primary difference from Cas9 is that it generates staggered breaks, also known as sticky ends. Cas12a...targeting experiments requiring single strand DNA generation, such as homology directed-repair. Cas12a is ...outlined below as well. Cas12a also has significant therapeutic promise due to the fact that it is smaller than...simplicity of Cas12a makes it easier to package for therapeutic delivery by AAV, which has strict capacity limitations...sequences of Cas12a to further optimize it for therapeutic and laboratory usage. Fast facts – PAM requirement...TTTN. Best for: homology-directed repair or therapeutic delivery.   Cas14 – Mini, but single strand ...

...such as p300 and CBP histone acetyltransferases or CARM1 methyltransferase. This attendant signaling feature...years evolved from chloramphenicol acetyltransferase to luciferase, the basic assay remains widely used...Both physiological processes are controlled by deliberate and compulsory actions instigated by the central...central nervous system signaling to peripheral effector organs, which then return information such as nutritional...communication of such signals between central and peripheral organs because of their established roles as ...available or being developed to treat a multitude of therapeutic indices, including hypertension, cancer, diabetes...action). Primary or core coregulators (those that interact directly with NRs) exist in steady-state complexes...

...challenge for CRISPR HIV-1 therapies is sequence specificity. Whereas antiviral therapies target HIV-1 at the...Today, HIV-1 can be controlled with antiviral therapies such that the virus is undetectable in the blood...demonstrated two potential strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription...usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma HIV-1, improving...binding sites increased activation of HIV promoter-luciferase constructs. In multiple HIV-1 latent T cell lines...the probability of off-target effects. In such a therapy, CRISPR/Cas9 would need to 1. excise the HIV-1 ...most HIV-1 patients. Obstacles for CRISPR HIV-1 therapy Both of these methods represent exciting advances...

...labs were also able to see movement and interaction between several different immune cells. These methods...to track cells from one organ to another. Thus several labs were able to decipher the journey of lymphocytes...low-energy photons (typically from the same laser) cooperate to cause a higher-energy electronic transition...molecule (see figure to the right). The excitation generated by these two-photons occurs only at a chosen focal...immune cells within organs and visualize how cells interact after a particular stimulus. Using an epifluorescence...quantitative measurements of cellular motility, interactions, and response dynamics. In the past 15 years...two-photon microscopy has uncovered host-pathogen interactions leading to the understanding of effector function...

...stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a...GeoCas9 is that it functions at temperatures much warmer than SpCas9 can tolerate. Harrington et al tested GeoCas9...at elevated temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure ... These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must...CRAA (R = A or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70...activity or longevity. GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs...GeoCas9 RNPs were incubated in reaction buffer at temperatures ranging from 25-80 °C. Activity is expressed...

...CRISPaint toolkit includes several donor plasmids for protein tagging, including luciferase (NanoLuc), fluorescent...lab developed a polymerase chain reaction (PCR)-based system that allows users to generate a plasmid containing...is expensive and time consuming to individually generate gRNAs and homology arm repair templates containing...scientists a convenient one tube assembly reaction to generate high-throughput targeting vectors. These vectors...includes a prebuilt donor plasmid library containing several types of fluorescent (EGFP, tagRFP, mCherry) and...donor plasmids from the library can be used to generate functionally unique repair templates. A final ... be easily modified to tag hundreds of genes, generating powerful genetic screening libraries. Additional...

...developed for each individual pooled library; generalized amplification protocols tend not to work as well... inserts of different sizes?), and the desired coverage for the library (How important is it that every...important factor to consider is your desired library coverage, since this will help decide what transformation...protocols aim for somewhere between 30x and 200x coverage, meaning that you need 30 to 200 times as many... your library. As an example, if you want 100x coverage of your library, and your library contains 5,000...thousands, if not millions, of plasmids, they are generally electroporated into electrocompetent cells to ...advantage is that chemical transformations are generally cheaper and easier. One library that Addgene distributes...

...human gene therapy. Molecular Therapy: The Journal of the American Society of Gene Therapy, 14(3), 316...the viral particle sneaks its way into cells. Generally, AAV serotypes have a primary cellular receptor...strains of one species, so be sure to check the literature and validate your choice of AAV vector for your...AAV1 was the first viral vector approved for gene therapy (Issa et al., 2023). It may be a good choice for...central nervous system, including neurons, AAV5 is generally the best choice for targeting astroglia. It has...rAAV treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes...AAV7, and AAV8 in the Mouse and Rat. Human Gene Therapy, 19(12), 1359–1368. https://doi.org/10.1089/hum...

... mutations, the risk is high. You are generating a gene therapy product with CRISPR to (hopefully) be ...experiments where they can’t be tolerated. Below we will outline several options to quantify these events... or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment which involves...-out edits in a gene(s) of interest and has an average off-target frequency of 5%. Your experiment involves...significant impact on your data. You are trying to generate a knockout cell line, with the goal of isolating... downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case...event efficiencies. In addition to these tools generating a prediction for off-target levels, most of them...

...including ours at Addgene, the overall purpose of a data pipeline is to perform several sequential computational...enabled us to rapidly iterate and optimize our pipeline. After reviewing and testing several platforms for engineering...parallels between Seqera’s mission and our own, as both address the need to accelerate science using different...sequencing at our facility using Illumina MiSeq Next Generation Sequencing (NGS) technology. As our repository...search for solutions led us to a partnership with Seqera Labs.              DNA sequencing drives our bioinformatics...sequences. More than half of these sequences were generated by Addgene as part of our QC process. Automation...other programs running on a computer, so it safely operates in isolation, and can be easily shared with others...

...survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....any fragments end-to-end without the exo/polymerase generation of annealed complementary regions. Why ...elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...restriction cloning), adaptor ligation for next generation sequencing (NGS) library construction, and circularization... does fidelity mean for a ligase? DNA ligases generally prefer fully Watson-Crick base-paired dsDNA substrates...as a correct base pair. DNA ligases have been generally found to have a higher discrimination at the upstream...

...through the biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in proximity...divisions).   Figure 4: Proliferation dyes can visualize proliferative capacity and speed of cells...research question and downstream application, one or several of those techniques can be used alongside standard...biotin-streptavidin binding and (b) antibody host species interactions. Created using biorender.com. Another way...perk is that one primary antibody can be bound by several secondary antibodies, increasing the fluorescence...are now stable for much longer, even at room temperature, allowing for a flow analysis to be shifted to...applications of dye stains in flow cytometry are cell proliferation and cell viability. Unlike intracellular labeling...

... these minor gRNA alterations can be exploited to quickly and inexpensively generate a potent Cas9-based...inactivate a gene or introduce a desired genetic alteration. Listen to Our Podcast Interview with Alex Chavez...Schematic of native (nuclease-competent) Cas9 interacting with either a full-length gRNA (20nt complementarity...from different bacterial species, each of which interacts with a distinct gRNA that allows users to decide...Although this is possible, this strategy suffers from several limitations. Perhaps most importantly, most Cas9...instance, when native (nuclease-competent) Cas9 interacts with a ≤15nt gRNA that contains an RNA adapter...induces potent gene expression with no genetic alterations observed at the target site.   Figure ...

...each, and potential delivery methods.   General considerations for planning your CRISPR experiment As ...Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts or knock-ins, or introducing mutations...PCR followed by gel electrophoresis and/or next-generation sequencing more appropriate? If you already...have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise for step 4, as this...system, a good first step would be to consult the literature to see if anyone has published a protocol that...enrich positive cells or a selection marker to generate stable cell lines. Transient or stable expression...positive cells. Likely contains a selection marker to generate stable cell lines. Packaging and Envelope plasmids...

...to detect this undesirable interaction. When non-specific interactions are suspected, use a preadsorption...controls for biological experiments, starting with general controls and then moving on to controls for antibody-related...interpretation of results. A positive control is generally a sample or group that will have a desired response...experiment above, you could repeat the tau western blot several times with the same samples and compare the results...results across runs. If the results vary considerably, then the high degree of variability in the process...antibody-only staining will allow you to see nonspecific interactions between the sample and the secondary antibody...-specifically to other molecules through the interactions of other antibody structures, such as the Fc...

...The general process is similar to standard immunofluorescence, but with some extra considerations (Im ... same considerations apply as for standard IF. However, there are a few specific considerations to keep...there is (quantification). But what if you’ve got several interesting protein targets and you want to know... in the cell? Or you know that the presence of several biomarkers together in the same tissue sample predicts...just what you need! How does it work? There are several ways to achieve multiplex IF. One of the most common...a good place to get started.  One important consideration when deciding whether you can do simultaneous...or conjugate with an enzymatic label such as horseradish peroxidase (HRP). Experiments using enzymatic...

...this reason I generally recommend that laboratories that have already attempted several CRISPR experiments... a part of a group (Biology of Host-parasite Interactions, Institut Pasteur, Paris) that changed genome... in highly similar content and the absence of iterative design; it is caused by not having the time to...to select a tool based on your project and not general opinion. I could spend a lot of time running through...functionality as well as how the user is expected to interact with the tool (a screen shot and overview of the...primer design are more and more common. User interaction: Software design elements that fall into this... category describe how the user is expected to interact with the software. This category is important ...

...first-generation bacterial ARGs (bARGs), used in bacteria, expressed poorly at body temperature (37oC)... transposase and outgrow the cells for a several generations to dilute the unintegrated plasmids (Fig....imaging was used. We set out to develop next-generation bARGs that could scatter ultrasound nonlinearly...Serratia sp. 39006. We next tested bARGSer with several inducible expression systems in E. coli. The arabinose-inducible...L-arabinose at 37oC for at least 12 hours, or at lower temperatures for longer. Cultures induced with arabinose ...  ARG expression in mammalian cells The first-generation mammalian ARGs (mARGs) enabled ultrasound visualization...in deep tissue. However, not unlike the first-generation bARGs, poor expression and acoustic properties...

... by the temperature parameters. For example, we found that changing the annealing temperature of the PCR...the composition of a viral vector solution, and generally these methods measure different characteristics...depending on the  experimental conditions used to generate AAV.  One study showed that approximately 50% ...preparations. Addgene has used this method to generally validate our viral vector preparation protocol...:  Absolute quantification by qPCR requires generating a standard curve of known concentration. We regularly...qPCR assays, we ensure that the standard curve generated is reliable, and can thus be used to accurately...Paolo, et al. "Systemic errors in quantitative polymerase chain reaction titration of self-complementary...