...is best for larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios...prevent the formation of replication competent lentivirus (RCL). Replication competence can arise from...end-users, size of the transgene, and downstream applications should all be considered when choosing the correct... as resources available, cost and downstream applications. For example, while the PEG precipitation method...

...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for CRISPR... therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a gene known...known as Crygc (Wu et al). They found that the cataracts phenotype could be rescued by zygote co-injection...substantial relief from both disorders. As of publication, submissions to the UK, EU, and USA federal drug...10.1186/s12943-022-01559-z Zarghamian P, Klermund J, Cathomen T. Clinical genome editing to treat sickle cell...

...). References 1. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-...methodologies, with a special emphasis on these five categories. Please note that many of the following cloning...relies on site-specific recombination reactions catalyzed by BP Clonase and LR Clonase enzyme mixes. After...there is no need for restriction digests, gel purifications, or ligations. The general Gateway cloning protocol...

...article, they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several...including excision or insertion, inversion, translocation or cassette exchange. By utilizing recombinases...BioRender.com In their recent ACS Synthetic Biology publication, Mary Dunlop’s lab generated a photosensitive...regions with Vivid (Vvd) photodimers. Upon the application of blue light, Vvd dimerizes bringing the C-terminal...

...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...Lower on-target activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very ... plasmid or group of plasmids' functions and applications. We hope that these articles make it easier ...FLIM (Fluorescence Lifetime Imaging Microscopy) applications. To demonstrate the power of mCyFP1, the Lin... Coselection efficiency is so high that some applications may permit working with edited “pools” of cells...showed high editing efficiencies with AsCpf1, indicating that ouabain coselection is a robust and flexible...be found at Addgene and in Figure S12 of the publication. Agudelo D, et al. Nature Methods. 2017. PubMed...

...technologies (Bradbury & Plückthun, 2015a).   Figure 1: (Top) Polyclonal antibodies are produced in animals...cells, for antibody production and subsequent purification.  Academic laboratories and companies have already...Edition. 2104. Cold Spring Harbor Laboratory Publications, New York. Helma J, Cardoso MC, Muyldermans ...

...most comprehensive for searching. Timesaving Tip #1: If you know the species that your sequencing result...search to check for expected ORFs, mutations or truncations. A primary advantage of blastx is that you do...other labs speed up their plasmid and cloning verification steps and free up more time for using your plasmids...

...genome engineering and microbiology.     References 1. Chen, Sean, et al. "Highly efficient mouse genome...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required...development. David Liu’s Lab has demonstrated the use of cationic lipid-mediated methods to deliver Cas9-gRNA RNPs... of eventually using this method for in vivo applications. This technique utilizes Cas9 proteins harboring...

... questions of how the brain operates. References 1. Callaway EM. Transneuronal Circuit Tracing with Neurotropic...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...like how our stem cells are housed in specific locations and give rise to progenitors during growth and...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...infection occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading ...limitations. First, these high levels of viral replication are toxic. Second, infecting subsequently higher...spread monosynaptically from these cells. These modifications take advantage of the fact that RABV is an enveloped...

...in restraint. The very first subheading in Chapter 1 asks “What is science?” That’s a very big question...drives and their ramifications. Readers unfamiliar with academia’s challenges with publication and funding ...Connecticut College who has been teaching and communicating science for over two decades, attempts to answer... primarily on fluorescent proteins and their applications, tackles much broader issues in this brief volume...sexism in science, problems with peer-reviewed publications and funding, bioethics in the age of CRISPR,... and her essential-but-unlauded role in the identification of DNA’s double helix. Perhaps to the dismay...dismay of middle-aged scientists, the identification of Green Fluorescent Protein (GFP) and its development...

... NGT PAM GAA PAM GAT PAM xCas9 3.7 41 (1) 32 (1.6) 13 (2.1) 38 (4.5) 7.2 (5.2) ...targeting anywhere in an exon. But things get more complicated if you’d like to use homology directed repair...They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase...test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that the clones...loci displayed a broad range of PAM sequences, indicating they may be less predictable than SpCas9 off-... that we have only scratched the surface of modifications that can be made to Cas9. It is a pleasant surprise...editing rates. This PAM flexibility should expand applications that require very specific targeting, like homology...

...Addgene with interests in education and scientific writing.     References 1. Martz, Colin A., et al. ... millions of people die from cancer-related complications each year. Despite these grim statistics, new...any of these genes through several different applications such as fluorescent protein fusion studies and.... "Systematic identification of signaling pathways with potential to confer anticancer drug resistance...

...for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the Addgene...emerging work by collaboratively discussing recent publications in a specific field(s). Most anyone who has ...the company at large, by polling and informal communications we made a decision to try out a Bioinformatics...major findings and then focusing more on broader implications rather than diving deep into the individual ....  These variations are suited for different applications and should be carefully chosen for each group...

... digging deeply into their data.     Fig. 1: Genomic fraction for each of five major ancestry ...Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry associated dependencies...machinery where to cut.      Fig. 2: Heatmap indicating loci where SNPs reside on sgRNA target sequences...Understanding the impact Fig. 3: Histograms indicating the frequency (x-axis) which SNPs map to thetargeting...same reference genomes and libraries. Further complicating the issue is the long and ugly history of reporting...

...cereblon, leading to rapid target degradation (Figure 1). Plasmids: We generated a series of plasmids that... and we will provide an update with vendor and catalog numbers. As we are happy to share aliquots of dTAG...

...effects (Zibitt et al. 2021).    Figure 1: A schematic illustration of CRISPR/Cas9-based approaches...it is important to carefully select the target locations of knock-in studies to avoid off-target effects...it comes to targeting precision and practical application. If the CRISPR/Cas9 system is not effective for...lncRNAs with high specificity, particularly in applications requiring smaller delivery systems (Zibitt et...

...dollar contracts behind closed doors. Fig. 1: The mythical land of negotiations.    You negotiate...fingers or Ethiopian?) to where you are going on vacation (New York City or Hawaii?). So how can you approach...been: A job offer conflicts with a pre-planned vacation I’m looking forward to. How can I negotiate my... I’m excited while still being able to take my vacation? I want to learn more about a different area of...

...editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) and targeted...and quiescent, post-mitotic mammalian cells. Applications of CRISPR delivery by adenoviral vectors Since...Strategies for Tissue-Specific Targeting. In: Applications of viruses for cancer therapy. Elsevier, pp ...Holkers M, Maggio I, Henriques SFD, Janssen JM, Cathomen T, Gonçalves MAFV (2014) Adenoviral vector DNA...

...impacts of neural circuits on behavior. Figure 1: An overview of the chemogenetics system. Why use...combined with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...

...necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes come in many.... Tracking samples - it's all about location, location, location There are many different types of samples...but we also have a dedicated and detail-oriented lab team that works carefully to catch mistakes. Each sample...assigning a physical location to your samples, include as much detail about that location as possible. Are...sample a unique location in a specific freezer will help ensure that samples are easy to locate in freezers...also help us keep track of the tubes’ physical location in the lab. These barcodes additionally allow ...Addgene. Each sample at Addgene has four layers of location which can all be extrapolated from the 2D barcode...