Gao, Y. et al. 5g). Robust expression of DsRed-Express2 in mouse bone marrow hematopoietic stem and progenitor cells. A second model used tdTomato to quantify breast cancer tumor growth in response to target gene activation (Johnstone et al. If the fluorescent protein has a strong tendency to form oligomers, it is more likely to alter or hinder the original function of the tagged protein. & Glick, B. S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Hence, the modality of repression can be defined as analog in the first scenario and digital in the second (Fig. Cai, Y. et al. Exposure time: 1 sec. NOT FOR USE IN DIAGNOSTIC PROCEDURES. HeLa cells were transiently transfected via a lipid-based method, with mammalian expression vectors encoding mCherry fused to either human cytoplasmic actin (Panel A) or tubulin (Panel B). A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs. Cytometry. A strain with inducible expression of mCherry that was transported to the periplasm was constructed (SI Appendix, section 1.2.2), and growth experiments demonstrated even distribution of mCherry throughout the periplasm and no migration to the cell pole in response to oxygen was observed (SI Appendix, Fig. 6f). The beads consist of a mixture of six distinct populations that vary in the number of attached AcGFP1 molecules, giving each population a distinct fluorescent signature. The peak representing the lowest intensity was adjusted to fall in the window between 10 x 100 and 5.0 x 110 . Plasmid and viral vector formats for gene function and expression studies involving red fluorescent proteins. Red: cells in G1 phase. It is however technically challenging, since it requires the ability to quantitatively modulate protein abundance. Auxin (3-Indoleacetic acid, IAA, GoldBio #I-110-25) was dissolved in EtOH to a 400mM stock dilution, according to the manufacturers instructions. This work was supported by the Max-Planck Research Group Leader program, E:bio Module IIIXnet grant (BMBF 031L0072), Human Frontiers Science Program (CDA-00064/2018) and ERC Starting Grant CisTune (948771) to E.G.S. Bintu, L. et al. For 5 selected concentrations we analysed target gene expression and the capacity of self-renewal after 3 days of dTAG-13 titration (Fig. All systems could be homogeneously titrated (tBFP), but again striking differences in the distribution of target gene repression (EGFP) were observed (Fig. Its high solubility leads to low cytotoxicity. A mESC line with Nanog fused with P2A-mCherry at its endogenous locus, stably expressing CasTuner (FKBP12F36V-hHDAC4-dCas9), was transduced with Nanog-targeting or non-targeting guides in the presence of dTAG-13. DsRed-Monomer is a true monomeric mutant of our red fluorescent protein fromDiscosomasp. Schulz, E. G. & Heard, E. Role and control of X chromosome dosage in mammalian development. Schulz, E. G. et al. Among the tested marker genes, Nanog was the only one that showed a non-monotonic dose-response curve (Fig. DsRed-Express and DsRed-Express2 are ideally suited for flow cytometry, with an extremely low level of green autofluorescence. We indeed observed induction of the TE program and loss of mESC markers, when Oct4 levels dropped below ~30% (Fig. (A) Compensation values are indicated, however, plots are displayed without compensation applied. The obtained graph shows that the bead suspension contains six well-distinguished populations with different fluorescent intensities. A.F.U. In parallel to dTAG-13 removal, the KRAB-Split-dCas9 cell line is also treated for 4 days with 100M ABA to induce tethering of KRAB to dCas9. To quantify protein abundance from flow cytometry data, the Median Fluorescence Intensity (MFI) was calculated for a given sample, followed by correction for the cells autofluorescence through subtraction of the MFI of one or multiple non-fluorescent control samples of the same cell line. The KRAB-Split-dCas9 plasmid was generated by inserting the FKBP12F36V domain C-terminally to dCas9 into the plasmid pSLQ2818 pPB: CAG-PYL1-KRAB-IRES-Puro-WPRE-SV40PA PGK-ABI-tagBFP-SpdCas9 (Addgene plasmid # 84241; a gift from Stanley Qi), after exchanging the Puromycin resistance with a resistance for Blasticidin. The ProteoTuner systems allow a protein of interest to be expressed as a fusion protein containing an extremely labile destabilization domain (DD). Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Nevozhay, D., Zal, T. & Balzsi, G. Transferring a synthetic gene circuit from yeast to mammalian cells. AcGFP1 is a monomeric protein. 2007),E. coli(Pradel et al. In addition to the tunable CasRx and hHDAC4-dCas9 systems of the CasTuner toolkit we again included the widely used KRAB systems and dCas9 alone for comparison. & Sawin, K. E. Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast. Nucleic Acids Res. The elution profile does not display higher molecular weight species and provides strong evidence that DsRed-Monomer is a true monomer. Genes Dev. Snaith, H. A., Samejima, I. CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression, $${dR}/{dt}={\beta }_{R}-{\alpha }_{R}\cdot R$$, $${dT}/{dt}={\beta }_{T}(R)-{\alpha }_{T}{T}$$, $$R(t)/{R}_{{st}}=1-{e}^{-{\alpha }_{R-}t}$$, \({t}_{1/2}=\,\frac{{{{{\mathrm{ln}}}}}(2)}{{\alpha }_{R-}}\), $$\,\frac{T(R)}{T(R=0)}=\frac{1}{1+{(\frac{R}{K})}^{n}}$$, $$T(t)/{T}_{{st}}=1+({T}_{0}-1){e}^{{-\alpha }_{T}t}$$, $$Target\,FC={T}_{\min}+\frac{{T}_{\max}-{T}_{\min}}{1+{\left(\frac{[X]1/2}{X}\right)}^{n}}$$, $$Target\,FC={T}_{\max}-\frac{{T}_{\max}-{T}_{\min}}{1+{\left(\frac{[X]1/2}{X}\right)}^{n}}$$, https://doi.org/10.1038/s41467-023-38909-4. Occasional delays may occur due to unforeseen complexities in the preparation of your product. Fluorescent proteins from nonbioluminescent Anthozoa species. Res. Terms and Conditions
We then used qRT-PCR to quantify the expression of NANOG target genes Esrrb, Rex1 (Zfp42), Nrb01, Sox2 and Klf4, which have been reported to be activated by NANOG42, and of Xist, which is known to be repressed by NANOG43 (Fig. In fact, when comparing repressor abundance (BFP) and repression strength (EGFP), hHDAC4-dCas9 appeared to be more potent than KRAB-dCas9 (Fig. Trends Biochem. Takara Bio USA, Inc.United States/Canada: +1.800.662.2566 Asia Pacific: +1.650.919.7300 Europe: +33. Monitor the activation of different promoters or promoter/enhancer combinations using traditional promoterless reporter constructs or next-generation on-demand reporters, which use ProteoTuner technology to provide extremely low background and bright signals. Open Access funding enabled and organized by Projekt DEAL. ZsGreen1 was used to label human estrogen receptor-positive breast cancer cells, and fluorescent whole-body imaging on living, unanesthetized, immunocompromised nude mice identified lymph node metastases that arose from estrogen-dependent tumors grown orthotopically in mammary glands. DsRed-Monomer has been expressed as a fusion with a large panel of diverse proteins with diverse functions and subcellular locations. Indeed, direct degron-tagging would be an alternative approach to homogeneously titrate protein levels, which would allow faster response dynamics compared to CasTuner, where the response time is limited by the repressor half-life, in addition to the mRNA and protein half-life of the target gene. Flow cytometer calibration beads for AcGFP1/EGFP and mCherry - Takara Bio Through our Takara, Clontech, and Cellartis brands, our mission is to develop high-quality innovative tools and services to accelerate discovery. Eukaryotic cells have the ability to quickly change their shapes, for example, while moving toward the site of an infection or during intracellular transport of macromolecules, membrane vesicles, and organelles. The authors declare no competing interests. Soc. Song, L., Han, M.-H., Lesicka, J. Panels C and F. Kernels from a segregating transformed line under white light and fluorescent light. For Xist, where NANOG binds together with multiple other factors including SOX243,44, repression might for example only be released, once NANOG and SOX2 are depleted, which only occurs at lower NANOG levels. At this setting, the remaining bead populations showed 5 distinct, well-separated peaks. Cell Stem Cell 11, 491504 (2012). Gilbert, L. A. et al. AcGFP1 has been widely validated as a fusion tag, for a wide variety of proteins with diverse functions and subcellular locations (Figure 1, Panel C). A disadvantage of the latter approach is a partial gene repression in the absence of effector domain recruitment, because dCas9 itself still occupies the target gene promoter19. Another example for a gene-dosage sensitive process is the differentiation of pluripotent stem cells into different lineages. Methods 5, 9557 (2008). (0)77.565.6999FOR RESEARCH USE ONLY. The dotted line shows the 99th percentile of non-fluorescent control cells. Fucci cell cycle reporter vectors deliver fluorescent, ubiquitination-based, cell-cycle indicators that allow you to identify cells in various phases of the cell cycle. A. 24, 36909 (2005). Bright fluorescent proteins make excellent reporters. Initially, the Timer fluorescent protein emits green fluorescence, but as time passes, the fluorophore undergoes additional changes that shift its fluorescence to longer wavelengths. Repression tunability can be verified only by single-cell measurements, since at the cell population level, bimodal or homogeneous intermediate distributions are indistinguishable (Fig. Monkhorst, K., Jonkers, I., Rentmeester, E., Grosveld, F. & Gribnau, J. X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator. As with other fluorescent proteins, the pFP-DR vectors are human codon-optimized and retain the same spectral properties as our standard fluorescent proteins. In these constructs, dCas9-tRFP and tBFP are transcribed together as a bicistronic unit, but are then translated into two separate proteins (Fig. Methods 13, 10431049 (2016). Having demonstrated tunability of CasTuner in mESCs, we decided to test the system in more differentiated human cells. FOR RESEARCH USE ONLY. Transfection efficiencies for mCherry mRNA alone . BrightComp eBeads compensation beads provide a consistent, accurate, and simple-to-use technique for setting flow cytometry compensation when using fluorescent protein-expressing samples. EF-1 alpha promoter vectors are useful in embryonic stem cells, where the CMV promoter has diminished activity in these cell types. To this end, we designed a sequence containing three 23 base pair-long guides interspaced by 2 optimised direct repeats, previously described66 and BsmBI restriction sites to produce compatible overhangs to the entry vector. The system employs the FKBP12F36V degron to precisely control Cas-repressor abundance by varying the concentration of the dTAG-13 ligand. AcGFP1 elutes from the column at a retention time corresponding to a molecular weight of 24 kDa. The "plus" end of each filament has a much higher affinity for monomeric actin than the opposite or "minus" end of the filament (Figure 1). An account with takarabio.com entitles you to extra features such as: Creating and saving shopping carts Keeping a list of your products of interest Saving all of your favorite pages on the site* Accessing restricted content. DsRed-Monomer has an excitation maximum of 556 nm and an emission maximum of 586 nm. mESCs=mouse embryonic stem cells; EpiSCs=epiblast-like stem cells; PE=primitive endoderm; TE=trophectoderm. But these techniques are limited to capturing the state of the actin filament network at the time of staining or labeling, and cannot monitor the dynamics of actin filament rearrangement. Primer sequences are listed in Supplementary Data1 part D. Cell samples for flow cytometry were harvested by washing with PBS, dissociation with trypsin for 7min at 37C and resuspension in serum-containing medium (DMEM (Sigma), 15% ESC-grade FBS (Gibco), 0.1mM -mercaptoethanol). A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs. Such dose sensitivity can arise from the need to maintain the right stoichiometry within a protein complex1. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters. The experimental distributions were log10-transformed prior to the test and the three replicates for each dTAG-13 concentration merged. The P2A peptide allows cleavage of (degron-)dCas9-tRFP and tBFP into two separate proteins. Moreover, we demonstrate the applicability of the system in studying the dose-dependent action of NANOG and OCT4 in controlling target gene expression and cellular phenotype. mCherry has been successfully fused to several proteins, including actin and tubulin (Figure 1). and E.G.S. Such a mechanism is also thought to allow spreading of the repressed state along the DNA to form chromatin domains, and to underlie epigenetic memory34,47,50. Takara Bio is proud to offer GMP-grade manufacturing capabilities at our award-winning facility in Kusatsu, Shiga, Japan. In many cases, oligomers can also be effective choices. For this reason, we took advantage of Lenti-X lentiviral delivery and ProteoTuner technology to develop a simpler alternative. So, the strategy I will follow is : 1. 24, 372376 (2000). Pseudonative gel electrophoresis yields a fractionation profile that is consistent with a monomeric protein, and similar to the monomeric green fluorescent protein AcGFP1. The approach is therefore relatively simple and cost-effective, allowing the study of how the quantity of different genes relates to a phenotype, thus preserving the scalability that characterise CRISPR-based technologies. In order to demonstrate the Gain Independent Compensation function, we compare the results from different scenarios in figure 3. All constructs were tagged with the FKBP12F36V degron at their N-terminus and with tBFP at their C-terminus, which allowed monitoring of repressor levels by flow cytometry. An example of the gating coordinates used for double sorting of cells with degron-Cas-repressor systems is given in Supplementary Data3. FOR RESEARCH USE ONLY. Far red proteins such as HcRed1, mRaspberry, and mPlum are preferred forin vivoimaging since they avoid the natural green autofluorescence produced by plant and animal cells. We sought to create a tool for tuning endogenous gene expression that can be easily applied to any target gene. Natl. The mean of three biological replicates is shown. Genet. 5e). DsRed-Express is a tetrameric protein that elutes at an earlier retention time (33 min) corresponding to a molecular weight of 89 kDa. 20 l of the mCherry Flow Cytometer Calibration Bead Suspension was thoroughly resuspended in 1ml of 1X Flow Cytometer Calibration Beads Dilution Buffer. Am. Without the addition of a functional promoter, this vector willnot express the Fluorescent Timer. For the dTAG withdrawal time course, where all repressors reached their maximal level in <25h, the mean MFI of the 25h150h time points was set to 1 and the mean MFI at time 0h0. Different approaches have been applied to make CRISPRa/i inducible, typically relying on conditional stabilisation or on inducible dimerisation between dCas9 and the effector domain22. =Arbitrary Fluorescence Units. J. Hum. An ideal degron domain would support a wide dynamic range of repressor levels and its complete removal, when not needed. Flow cytometer analysis of AcGFP Flow Cytometer Calibration Beads. 20, 837 (2002). Biotechnol. Cell Biol. 1bg). DsRed-Monomer is soluble and displays even, consistent, and homogeneous distribution in HeLa cells. Nature Communications Genesis 45, 593605 (2007). Study promoter activity using the Living Colors Fluorescent Timer, a fluorescent protein that shifts color from green to red over time (Terskikh et al. Biol. Here histone-modifying enzymes are recruited through binding to the modification they deposit either directly or in an indirect manner through additional proteins. 3e). 5c, Hall-York test for unimodality, p<0.01, see methods for details). Therefore, we named these two systems collectively the CasTuner toolkit. Since its application is much easier than genetic perturbations, which have previously been used to investigate Oct4, our system will facilitate the study of dose-dependent effects for a larger number of factors in the future.