... RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix, purple.   Apo Cas9 changes conformation...viewed from the same angle to highlight conformational differences. HNH, red; RuvC, orange; CTD, yellow; REC...resolved due to structural disorder. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix...and are important for robust activity in cells (Jiang & Doudna, 2017). Within an interior cavity, Cas9...Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing with a complementary DNA strand...state (PDB ID 4ZT0, bottom), seen from the same angle. Box highlights the DNA-binding region of the CTD...and absent from that structure. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix...

...Novartis researcher, Carrie Bhang. The ClonTracer Library, deposited by Carrie Bhang, a research investigator...in Frank Stegmeier’s lab in Novartis Oncology.  Bhang did her PhD work at Johns Hopkins and came to Novartis...The ClonTracer library originally came about as Bhang and co-workers were trying to develop a way to quantify... with lentiviral pooled shRNA libraries. Later, Bhang says, “When we developed this tool, there were so...Using ClonTracer to study drug resistance Although Bhang and co-workers started using the tool to look at...cancer development, metastasis, and drug resistance, Bhang says “the [drug] resistance story panned out quickly...recent Nature Medicine paper. In the publication, Bhang and coworkers used the ClonTracer library to determine...

...amplification. Zhou S, Feng S, Brown D, Huang B. PLoS One. 2020 Bo Huang Yellow Venus pBiFC-VN173, pBiFC-... with split fluorescent protein tags. Tamura R, Jiang F, Xie J, Kamiyama D. Commun Biol. 2021 Daichi Kamiyama...Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques. 2006 Chang-Deng Hu Green sfGFP Split super-folder GFP, our...fluorescent protein. Kamiyama et al. Nat Commun. 2016 Bo Huang spGFP Split superpositive GFP Split-superpositive...protein labeling. Feng et al. Nat Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use...Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques. 2006 Chang-Deng Hu Venus Improved N-terminal fragment VN155...

...concentration lies within the dynamic range of the curve. The dynamic range is the linear span between the lowest...the dynamic range of your standard curve, so if your sample does not fall within this range, it may be ... of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. The specific dilution series used...Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual step of the series...final readout. When preparing a standard curve, change pipette tips and mix thoroughly by inversion or... how well the data points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating...that the curve can accurately measure.  Dynamic range depends on a number of factors, including the dilution...

...that surround changes in gene expression and determine who’s a key driver of these changes. By generating...on gene regulation. References Han, J., Zhang, Z., & Wang, K. (2018). 3C and 3C-based techniques: the..., X., Chen, Y., Zhang, Y., Liu, Y., Liu, N., Botten, G.A., Cao, H., Orkin, S.H., Zhang, M.Q., & Xu, J....and proteins that forms chromosomes. These long range DNA interactions are often detected with chromosome... CAPTURE method, the Xu Lab made the following changes to create CAPTURE 2.0:  Replace the biotinalatable...control gRNA or predicted gRNA off-target sites. Long-range chromatin interactions identified with the multiplexed...of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions A third application for...

...heterogeneity, and/or cell position changes (To et al., 2016, Zhang et al., 2019). Thus to effectively ... 27447051. PubMed Central PMCID: PMC5026494. Zhang, Qiang, et al. "Designing a green fluorogenic protease...site. Cutting the linker by a caspase results in a change in fluorescence emission. FRET and fluorescence..., and thus quantify, due to small fluorescence changes of the donor and acceptor after proteolytic cleavage...need a protease reporter that has a large dynamic range and high brightness. The Shu Lab from UCSF designed...However, the lab wanted to increase the fluorescence range to better study the spatio-temporal dynamics of ...fluorescence 100-fold compared to other reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains...

...dilution range from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from ...of interest to understand the dynamic or working range of a given reagent (usually an antibody).    ...concentrations are within the antibody’s dynamic range. Created with biorender.com   Alternatively,... protein aggregation, you may want to consider changing your incubation temperature. Some proteins aggregate...kDa) and a 0.45 µm (p) membrane for larger ones. Changing your membrane size is probably the easiest troubleshooting...antibodies come with a recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary...recommended dilution. If the antibody dilution’s dynamic range encompasses your protein’s expected concentrations...

...tools needed to do so. Cepko lab researcher Jonathan Tang wanted to express a variety of proteins in single...develop the mouse lines required for this purpose. Tang wondered if he could use previously established ...selectively manipulate only GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies...Envisioning GFP as a substrate promoting dimerization, Tang et al. tested pairs of their GFP-binding proteins...transcription factor, activating luciferase transcription. Tang et al. coined this system T-DDOG (Transcriptional...GFP). In testing the specificity of their system, Tang et al. found that YFP and CFP can activate certain...different promoters. Moving to an in vivo system, Tang et al. electroporated T-DDOGs, GFP, and a reporter...

...with 5’ overhang TGGA and 3’ overhang TCCG can be ligated into a vector containing those overhangs. Entry...sequence, creating four base flanking overhangs. Since these overhangs are not part of the recognition sequence...targeting. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove...destination vector contains sites with complementary overhangs that direct assembly of the final ligation product...Entry DNA overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification...Gate cloning is its scalability. Unique 4 base overhangs can be used to assemble multiple fragments - up...commonly assembled in a single reaction! These overhangs specify the desired order of fragments, and the...

... #1, any change in the normalized intensity of your POI could reflect either a genuine change in your ...lies the linear detection range. Figure 4: The linear detection range of the assay is between the...detection range, band intensity measurements become extremely susceptible to error, since tiny changes in intensity...your POI, but simply whether its concentration changes in response to experimental parameters. This is... even hit the gel. But what if your experiment changes the expression of your loading control without ...your POI or an opposite change in your loading control. Assumption #2: The loading control can be accurately...control points are outside of the linear detection range. As a result, they have almost the same value on...

...switchable (e.g. rsEGFP, Dreiklang). Fluorescent Timers (FT): These proteins change their color over time...example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent Sensors: These FPs change their excitation/emission.../em spectra (e.g. mKeima) or change fluorescent intensity upon pH changes (e.g. pHluorin, pHTomato). ...classified by the emission color (emission wavelength range) FPs are usually classified by emission color as...as outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP...nm Green 499 - 519 nm Yellow 524 - 538 nm Orange 559 - 572 nm Red 574 - 610 nm Far-Red 625 -... ≥ 670 nm In addition to emission wavelength range, there are other traits that need to be considered...

...cells expressing the optogenetic tool causing a change in cellular activity or gene expression. Experimental...directly open and close when exposed to light and change the membrane potential. OptoXRs are modified GPCRs...activate downstream signaling pathways that can change both the membrane potential and a host of other...engineered molecules that induce conformational changes with light stimulation to either activate or inhibit...activity upon exposure to light within a specific range of wavelengths. Excitatory channels allow positively...When exposed to light, there is a conformational change in the extracellular domain which activates the...the binding pocket of an engineered receptor that change shape with light exposure to activate them. PORTLs...

...flank the desired change with 4-5 silent changes in the wobble codons, i.e. make changes to the third base... translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds- ...sequences, point mutations or other small base pair changes, and the addition of protein tags. It also has ...to restriction enzyme cloning where the genetic changes occur in a test tube. The donor DNA substrate only...partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough, a ssDNA whose...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...prone to mutations and will have more unintended changes to their genomes. 2) Designing ssDNA oligos that...

...base editor precisely changes a single base with an efficiency typically ranging from 25-75%, while the...2018): 627-630. PubMed PMID: 29476916. 10. Kang, Beum-Chang, et al. "Precision genome engineering through...base precisely? Until 2016, precise single base changes were only possible through exploiting the homology-directed...the success of precise change via HDR limited to 0-5%. This blog post covers a brief review of different...wonder if a CRISPR-base editor can perform this change. However, there are no naturally known 'DNA adenosine...lack of base editors for plant systems. Thus, the Yang Lab at The Pennsylvania State University (PSU) is...hAPOBEC3 deaminase can edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong...

...poor dynamic range, meaning that if your blot contains one protein in the femtogram range and another ...dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic range for fluorescent...to switch from one readout method to another or change primary antibodies as needed. However, more antibodies...especially if you need a specific conjugate, and changing your reporter molecule requires either re-conjugating...protein(s) of interest.  Determining linear detection range If you are quantifying your western blot data, you... you’ll need to determine the linear detection range of your protein. The most common way to do this is..., quality, and stability Limited protein size range Tris-acetate 40–500 kDa Tris-tricine 150...

...recognition sequence, creating flanking overhangs. These overhangs can be customized to link together multiple... 2016 and last updated Sep 10, 2020 by Jennifer Tsang. CRISPR makes it easy to target multiple loci - ... editing or labeling efficiency doesn’t usually change when you add multiple gRNAs on one plasmid. Sound...appropriate Type IIS enzyme, the unique flanking overhang sequences can link together to allow for ordered... Figure 2: gRNA target sequences (colored rectangles) are cloned into various plasmids using oligonucleotides...constructs. When these plasmids are digested, unique overhangs (here, O1-4) adjacent to the cut sites “link” ...This step is necessary to generate all of the overhangs needed for the final ligation step. Plasmids are...

...science (from climate change deniers to anti-vaxxers), and the very real dangers of quackery, which is...scientists to change their priorities. For many scientists, COVID-19 will fundamentally change what they ...described in Chapter 3, and of course the rapidly changing world of genome engineering via CRISPR.  Though...non-scientists, Zimmer’s observations will likely range from mildly surprising to genuinely shocking. Mainstream..., and posits the way science is being done has changed significantly over the last decade. He describes...adoption by the scientific community and their game changing nature. Full disclosure: Zimmer also spends some...played in these fields, and seeing names like Feng Zhang, Jennifer Doudna, Karl Deisseroth, and Ed Boyden...

...detect nucleic acids at the atomolar level. The Zhang lab has adapted natural RNase activity of the Cas13...Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase (Abudayyeh et al 2016...specific reporter and the activation of Csm6. The Zhang lab showed that, by adding Csm6 and a Csm6 specific...Applications of the SHERLOCK detection system The Zhang lab demonstrated that SHERLOCK could reliably distinguish... in the field (Myhrvold et al., 2018). Now, the Zhang lab has shared a protocol for using SHERLOCK to ...undergoing validation studies.     Jennifer Tsang contributed to updating this article. References... L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F (2016) C2c2 is a single-component programmable...

...conformational change in the GPCR causing the activation of the Gα subunit. The activated Gα exchanges bound GDP...PRESTO-TANGO (parallel receptorome expression and screening via transcriptional output - TANGO) (Kroeze...Kroeze et al., 2015). PRESTO-TANGO uses a modified Tango beta-arrestin recruitment assay to measure receptor...Sassano MF, Huang X-P, Lansu K, McCorvy JD, Giguère PM, Sciaky N, Roth BL (2015) PRESTO-Tango as an open-source...examples with plasmids deposited at Addgene.  PRESTO-TANGO: The ligands that activate many GPCRs are still ...for unknown GPCRs. You can find the entire PRESTO-TANGO kit at Addgene! TRUPATH: Bryan Roth’s lab also assembled... internalization of two different receptors- angiotensin II type 1 receptor and the β2 adrenoceptor. ...

...most common diseases. References Zhang Y, Yin C, Zhang T, Li F, Yang W, Kaminski R, Fagan PR, Putatunda...publication at Addgene. Wang Z, Pan Q, Gendron P, Zhu W, Guo F, Cen S, Wainberg MA, Liang C. CRISPR/Cas9-Derived...integrated HIV-1 genome. For a targeted approach, Zhang et al. turned to the CRISPR/Cas9 Synergistic Activation...and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target the 5’ ... in the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed that most of...Shortly after Kaminski et al. published their results, Wang et al. showed that HIV-1 could escape from CRISPR...mutations were located near the Cas9 cut site, leading Wang et al. to conclude that some Cas9-derived indels...