...base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated with...Lower on-target activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very ...cassette via Cre recombination. Each knockout includes unique barcodes and universal priming sites to ensure ... the Chi-Bin Chien lab (Kwan et al., 2007) and includes four promoters, six fluorophores with nonoverlapping...The set of CyRFP constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression...codons) with kanamycin cassettes and were carefully designed to allow for easy removal of the resistance cassette...essential in B. subtilis 168. Each individual strain includes a xylose-inducible dCas9 paired with a single,...

...for RaPID is an RNA motif of interest. See figure 1 below for details on the key components of RaPID. ...novel RNA-protein interactions, Ramanathan et al. designed an RNA bait containing three well conserved but...

...Set a goal for paper-reading, even starting out at 1 paper/week will be useful. Check out the hashtag #365papers...down your own results; are they the same as the description the authors give? Ask yourself if the results...

... to the use of multiple antibodies. Figure 1: Overview of using Sandwich ELISA to quantitatively...ELISA is a modification of the sandwich ELISA as described here, the direct and indirect ELISA (more details...

... digging deeply into their data.     Fig. 1: Genomic fraction for each of five major ancestry ... reference?  The CRISPR guides used in the screens they analyzed were designed to have a perfect match...African descent have approximately 20% more single nucleotide polymorphisms (SNPs) that map to guides in the...accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’ genomes...modify the CRISpick algorithm to prevent guides from being designed that target regions with high genomic...coming through an unexpected source: the CRISPR guides, short pieces of RNA that bind to a cell’s DNA,...x-axis) which SNPs map to thetargeting sequences of guides across 8 CRISPR libraries. Samples are divided ...

...started:  Subscribe to Career Blog Posts from Addgene 1) Twitter – Get your voice out there Here’s one thing... Guardian, BBC Breakfast or the Londonist. BBC provides a peak into their day-to-day business with popular...Co-Ordinating Centre for Public Engagement (NCCPE) provides on-site courses in public engagement for institutions...

...viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University...plasmids, especially those of the CRISPR variety (see slides from my talk below). Largely organized by Dmitry...CRISPR regions of the prokaryotic genome. Then he desribed how P. furiosus has systems to defend against ...

...treatment and improving patients lives. References 1. Ong-Abdullah, Meilina, et al. "Loss of Karma transposon... of the DNA with a high frequency of C and G nucleotides). The conversion of cytosine to 5-methylcytosine..., which binds selectively to lysine-methylated peptides. This construct is in the middle of a FRET pair...

...in restraint. The very first subheading in Chapter 1 asks “What is science?” That’s a very big question... Zimmer provides several. In Part Three of the book, called simply “Old Science,” he describes the now...teaching and communicating science for over two decades, attempts to answer it in five paragraphs, with...recent resurgence of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing... science.”  The three chapters in this section describe the challenges of trusting science in a “fake ...changed significantly over the last decade. He describes the detection of gravitational waves--a story ...collaboration from scientists around the world. The paper describing the waves’ detection had over 1,000 authors. ...

...impacts of neural circuits on behavior. Figure 1: An overview of the chemogenetics system. Why use...DREADDs DREADDs DREADDS, or designer receptors exclusively activated by designer drugs, are G protein-coupled...Relationship Studies for Designer Receptors Exclusively Activated by Designer Drugs. ACS Chemical Neuroscience...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity...Roth's and Takafumi Minamimoto's lab called deschloroclozapine (DCZ) shows promise as a CNO alternative ... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nature...

... NGT PAM GAA PAM GAT PAM xCas9 3.7 41 (1) 32 (1.6) 13 (2.1) 38 (4.5) 7.2 (5.2) ...insertion site, the corresponding gRNA might be undesirable due to potential off-target sites or low predicted..., resulting in lower overall genome frequency. Despite the progress we’ve made, there is still a need ...bacterial polymerase subunit ω. This construct is designed to activate transcription from the accessory plasmid... stringency and promoting survival of only the broadest-targeting variants. For the final round of evolution...spectinomycin resistance. xCas9 3.7 showed the broadest PAM range, recognizing NG, NNG, GAT, and CAA PAMs...scaffold. With an NGG PAM, xCas9 3.7-VPR constructs modestly outperformed SpCas9-VPR in activating transcription...

... questions of how the brain operates. References 1. Callaway EM. Transneuronal Circuit Tracing with Neurotropic...widely used in neuroscience. In this post, I’ll describe how rabies virus (RABV) can be used in the brain... by modifying the wild-type RABV genome, which encodes a single envelope protein called rabies glycoprotein... neurons integrated into the brain over time (Deshpande et al., 2013). The study found that at early time...Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, Lepier ...Pubmed PMID 26190977. 6. Osakada F, Callaway EM. Design and generation of recombinant rabies virus vectors...

...target DNA quickly and accurately.  Figure 1: Steps in PCR reaction   This process of denaturation...addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique...polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents and a series of heating...protocol page, protocol video and reference on how to design PCR primers.  Types of PCR Since the invention ...  Digital droplet PCR or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration...Polymerase Chain Reaction (PCR) Protocol: How to Design a primer  Molecular Biology Reference Molecular...

...(RBD) of the viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing...library, each plasmid encodes the mutated spike protein and a barcode, and is designed so that the SARS-CoV....1074/jbc.RA120.01289     Novel protein cage provides simple enzyme protection in yeast bioproduction...enzymes in a reaction pathway, the MPyV system provides a simple, efficient method for protecting a single...- Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by anhydrous...

... education and scientific writing.     References 1. Martz, Colin A., et al. "Systematic identification...die from cancer-related complications each year. Despite these grim statistics, new strategies for attacking...cancers, especially those of the pancreas and colon. Despite the prevalence of Ras mutations in a variety of... enabled researchers to begin discovering and designing molecules that are showing some promise in their...in the screenshot below). Currently, the page includes interactive maps for the Ras, mTor, and p53 pathways...

...genome engineering and microbiology.     References 1. Chen, Sean, et al. "Highly efficient mouse genome...transfecting cells with a plasmid, such as PX459, which encodes Cas9 and contains a site for inserting a custom...targeted approach using RNPs has recently been described in vitro, with the hope of eventually using this...expertise. Final thoughts The Cas9-gRNA RNP method provides scientists with an additional means to deliver...other CRISPR delivery techniques Find tips on gRNA design Resources on Addgene.org Browse all CRISPR plasmids...

...for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the Addgene... on single papers could be more important.  Any slides, presentations, recordings, and other materials...those who were unable to attend. We capture the desktop and audio recordings of the meeting using teleconferencing... ensure that the journal club is useful for the widest audience, you should get feedback from those who...served. I hope that with some of the information described above you feel empowered to start your own journal...

...off-target effects (Zibitt et al. 2021).    Figure 1: A schematic illustration of CRISPR/Cas9-based approaches...non-coding RNAs (lncRNA). lncRNAs are over 200 nucleotides in length and lack an open reading frame, so ...transcription machinery of a target gene or DNA that codes for ncRNA. CRISPRa, on the other hand, recruits ...which targets RNA instead of DNA and selectively degrades RNA molecules. This system is often used to knock...

...which you’ve already fluorescently labeled. Figure 1: Using Split-BioID to Identify Protein Protein Interactions...specific protein-protein interactions, SplitBioID provides a candidate list of proteins that are part of ...

...article, they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several... Parts. This library delivers 2 randomly paired guides per construct and allows a reduced scale in screens...