...ll need a few controls specific to flow as an application. These controls will allow you to distinguish...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...commercial antibody pages. Flow cytometry can be a complicated and daunting process. Controls like single color...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...neurons and specific behaviors. Chemogenetics uses genetically engineered receptors to interact with small molecules... to optogenetics but instead of light, these genetically modified receptors are activated by small molecules...some GPCRs further to respond to specific, pharmacologically-inert, small molecules. These new receptors...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...

... light-mediated? The researchers split the proteolytically active light chain of BoNT/B into two fragments...that undergo light-induced dimerization. Upon application of blue light, iLID undergoes a conformational...rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue performance,...the field. Arlene Sharpe’s group at the Harvard Medical School developed a solution: CHIME, a CRISPR-Cas9...

...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines already expressing Cas9...

...of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism... for example).  Given that the liver is an immunologically active organ, this advance is likely to afford...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...clonal multiplexing of cells in live imaging application. Each visual barcode consists of a fluorescent...

...α-complementation. The system described above was put to practical use in the following way. Scientists engineered...insert. All colonies on this plate should be blue, indicating that your IPTG and x-gal are working as they ...of false positives: Blue-white screening only indicates the presence of AN insert, not necessarily YOUR... Jessica Welch...

...missing or damaged nucleotides that are common to biological sources of DSBs, and which need to be processed...generated in the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There.... NHEJ can also be engaged by variants of the canonical Cas9 approach. A pair of CRISPR guides that flank...Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular Cell 55:829...

.... Listen to our podcast interview with Harvard Medical School researcher Connie Cepko to learn about her...mentoring programs. The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet...This is a chance to practice leadership and communication skills. The leader also provides materials such...present formally often enough) and give honest, critical feedback for improvement. Go to a science or career...

...produce genetically modified cells in less than a month's time. Addgene: What are the implications of this...Musunuru: The kit includes 832 plasmids containing prefabricated pieces that allow you to build a DNA-binding...diseases. We are knocking out genes that have been implicated in disease or, in cases where particular variants...

...time it takes to make a plasmid could increase dramatically if you are piecing together several PCR products...to make plasmids that already exist, and we significantly reduce costs of producing plasmids with high-throughput...The faster methods, like Gibson assembly, are typically more expensive, so you’ll have to find a balance...calculations for any of the numbers in this post is a mathematical story well beyond my ability to tell. But even...

...protein expression in mammalian cells: easy to use biological tools are abundant! In this post, we will review...that! CRISPR interference (CRISPRi) targets a catalytically dead Cas9 fused to a transcriptional repressor... protein, then these systems need to be site-specifically knocked in. This can be labor intensive and ...and target-specific protein degradation. Nature Chemical Biology. May 2018. 14(5): 431-441. PubMed PMID...

...our second example we’ve added some simple but critical logic. Some steps fail “hard”, they reset the ...interchange them easily as needed. In this case, the physical considerations of the laboratory make object composition...science_name`. You'll need to decide given the technical details of the problem you're solving, but consistency...interesting biotechnology code.There are valid technical reasons to choose a non-Python stack, but if you're...

...donating and receiving labs)” — Jim Woodgett, biomedical researcher at Lunenfeld-Tanenbaum Research Institute...reagents and protocols so easy!” — Nina Jain, Harvard Medical School. We want to give a special thanks to the...decade. We did this the old-fashioned way: through physical outreach, visiting organizations, knocking on ...standardized agreements and electronic signatures to dramatically speed up MTA approvals. In 2013 we were thrilled...

...choose well. What are their qualifications? Do they have certifications? How can they demonstrate their...help you. A good coach usually will be a clear communicator and make direct suggestions for improvement ...she is especially excellent in coaching for communication skills. Industry transition is also an area ...

...search algorithm rather than pre-designed sets, so identical searches can return multiple answers. You might...of a lac operon dropped to 49% fidelity, with significant lot-to-lot variation. Additionally, this assembly...assemblies are possible, the two sets of data together indicate that 35-pieces is a reasonable upper limit for...Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology. https...

...-humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 ...ecotropic or amphotropic MuLE lentivirus provides significant genetic power and allows high throughput genetic...be used to construct complex lentiviruses for modification of mammalian cells with a single viral infection...Gammaretroviral Vectors: Biology, Technology and Application. Viruses. 2011. PubMed PMID: 21994751. PubMed...

...engineered microbes, used coffee grounds could be economically repurposed as starting material for the bioproduction... FLINC biosensors for live-cell imaging of biochemical activity Article contributed by Angela Holmes... live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence ...peak of 519 nm. These proteins have improved photophysical properties and can be used in quenching, FRET...

...its design changes for every new prime editing application. And optimizing its sequence is important, as... gRNA (Anzalone et al., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., ...other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com...structured motifs like mpknot, use the pegRNA Linker Identification Tool (pegLIT) to create linkers that minimize...

...ampicillin resistance cassette and an origin of replication, which are not schematized. Sizes of fragments...validated and were chosen because of their broad applicability in different model systems. What’s the error...five months. When they tested those clones (in duplicate), 93% had the correct banding pattern in the restriction...correct. Because they created the assemblies in duplicate, they were able to confirm that at least one plasmid...

...and should be varied as it can have important implications for the fusion protein effectivity), and a fluorescent...to visualize DNA, an editor targets DNA (via catalytically inactive dCas9) to modify chromatin. A fluorescent...both spatiotemporal protein degradation and quantification of this depletion using imaging-based measurements...BioTag, which can be used in protein affinity purification studies.    Duong et al., Genetics 2021. https...

...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... Jesse Zalatan’s lab deposited plasmids for a chemically inducible CRISPRa system in yeast is mediated... channelrhodopsin (C1V1) targeting parvalbumin cortical interneurons is now available as a ready to use...

...can contain different neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin... show a high proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in ...in high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...Hybrid for Multiplexed Editing and screening Applications (CHyMErA) Knockout Libraries express hybrid ...

...methods greatly improved CRISPRa, expanding its practicality and popularity in research (Tanenbaum et al....themselves. SunTag, SAM, and VPR have all shown significant improvements upon the initial dCas9-VP64 method...Performance Generally, VPR is found to have significantly higher activation levels than the initial dCas9... (2014) A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging....

..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...viral particles that have the gene deletion could replicate when challenged with the S. islandicus CRISPR-...Hematopoietic Stem Cells. Molecular Therapy - Methods & Clinical Development 9:390–401 . https://doi.org/10.1016...B, Bondy-Denomy J (2020) Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies....

... first at Brigham and Women’s Hospital/Harvard Medical School and then Boston University (institutions...committees would not function without long distance communication and learning to work with people despite the...the distance improved my verbal and written communication skills. Volunteering on these committees was ...Chair then stepped down, I became interim Chair automatically as it was an understood expectation of any Vice-Chair...

...detect explosives and rapidly indicate their presence. They are genetically modified organisms, and GMOs...things more interesting, we are working in a new technical discipline that nobody understands (synthetic ...that survived the literature review, survived technical feasibility analysis, and was a marketable product...

...These impurities cannot be removed during the purification step because they are inside the virus itself...sequence (gene). In a clean sample, there are typically fewer than 10 hits to a given sequence, and we...Guerin et al., 2020).  In addition to genome identification, we developed custom open-access Python scripts...control is recommended but not yet required for clinical-grade materials, and is not commonly performed...

...your gene of interest is difficult to target specifically with a gRNA). Be sure to keep these techniques...sequences are exchanged between two similar or identical molecules of DNA. Gene targeting takes advantage...homology is required, but 6 to 14 kb of homology is typical for targeting constructs. In the example shown ...cells participating in development or other physiopathological events to which the chosen promoter responds...

...genome editing technology that allows precise modifications of DNA (replacements, small insertions, and ... Liu, 2023). This method uses a prime editor (typically a Cas9 H840A nickase fused to a reverse transcriptase... design of the pegRNA has been shown to play a critical role in the editing efficiency of prime editing...configurations can be used to introduce specific target modifications. Since this means we could design hundreds to...

...to the same principles listed here. It employs biological best practices. For example, it ranks guides ...with NGS data were: Crispresso CrispRVariants AmpliCan We chose to adapt Crispresso by Luca Pinello ...HDR. Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide...published on how to do it best. Scientists are typically advised to mutate the PAM to which the guide binds...

...setup. For instance, BV510 and AmCyan have almost identical emission spectra but may be called BV510 on one...diagonal population, this is a sign of spillover and indicates that it is necessary to apply a compensation value...shift towards the X axis to become a perpendicular vertical line.     In general, most spillover between samples...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...their fused protein under certain conditions (typically the addition of an exogenous small molecule). ... size from several kDas up to ~35 kDa and are typically knocked-in via gene targeting or cloned in-frame...proteasomal degradation. Adapted from Nabet et al., Chemical Biology, 2018.    HaloTag The HaloTag was ... F-box protein must be expressed (inducibly, ectopically, stably… it’s your call). The TIR1 F-box protein...

...easier because individual labs will not need to ‘domesticate’ all the basic and necessary elements in order... you are in the lab. There is no PCR, no gel purification; the whole cloning process is so simplistic ...tweet about plant synthetic biology, science communication, women in science, and many other topics. What...well-rounded human beings. It’s also a great communication tool for work. Most Sainsbury scientists are...

...not the case for knock-ins. Knock-in mutations typically require an exact DNA sequence to be incorporated...know that HR is often mutated in cancer cells, specifically in breast and ovarian lines commonly used in...components of both pathways have been shown to be efficacious for increasing knock-in events (Yang et al., ...nutrients in their media, they will not undergo DNA replication, so make sure you are keeping an eye on those...

..., confirming again that sgRNA design plays a significant role in library efficiency. Try the Dolcetto ...lowly expressed genes or genes that are more drastically affected by gene activation rather than depletion...Calabrese pooled library and lentiviral prep here! Applications for the Root and Doench CRISPR libraries Overall...et al. "A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...manuscript to get reviewed and edited prior to publication, and this will delay the dissemination of your...bring your phone into the lab? Maybe having a dedicated lab-use tablet? The software also makes it easy...2,500 protocols covering a variety of bio and biomedical topics. Protocol Online Website: http://www.protocol-online.org...Freedom: The Expansion of Data Sharing in Research Publications by Jim Woodgett        ...

...resources you’ll need ahead of time and mute your notifications during. (Boyes, 2019) If you’ve taken the time...it down until you have something tangible and applicable to your situation.   Thinking Outside the Box...frequent, but establishing a few minutes every month dedicated to thinking about the future will improve your...community is supply chain shortages. When flasks, conical tubes or pipette tips are backordered for months...

... defective gene, Xm, the sequence of which is identical to the correct gene, Xw, except for a single base...precisely changes a single base with an efficiency typically ranging from 25-75%, while the success of precise... in the DNA by cutting only a single strand, dramatically reducing the likelihood of detrimental indel...known 'DNA adenosine deaminases' which would theoretically give rise to an adenine base editor. A massive...

... from depositing laboratories and associated publications, but our quality control (QC) standards have...In Addgene’s early days, we would ‘spot-check’ critical regions of plasmids deposited with us via Sanger...such as 96-well microplates, and thanks to our dedicated product and development teams, frequent updates...can be easily shared with others without any complicated setup. We created, tested, and debugged each ...

...without their problems, but much genetic and physiological data have been accumulated over the years using...check out our Mouse Modeling blog posts: Part 1: Genetically Engineered Mice and Part 2: Breeding and Crossing...nice to your microinjectionist, who has the strategically important job of delivering the payload! Last...interested in using CRISPR to develop therapeutic applications that better human health.     References Cong...