...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...

... 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...

...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...

...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...