...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...

... Sternson SM. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...a targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003):...cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution...

... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...of plasmids required for each input/output system; 2) removing all chemically inducible promoters; 3) improving...

...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially... roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...receptor constructs with AAV. In the case of channelrhodopsins, fewer channel proteins are required for the...

...Activation of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission from...mouse to deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that ...

...M. Sternson. 2008. “A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range...FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) and synaptic...PMID: 18614669. PubMed Central PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart....The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl...

...typically, normal (bell curve) or skewed (see Fig. 2). Assumptions – characteristics of a data set; ...levels of variance among data.     Fig. 2: Examples of data distribution profiles.   Parametric... positive is much higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise ...statistical facets of reproducibility. Adv Physiol Educ. 40(2): 248-252 (2016). 0.1152/advan.00042.2016 Finch, W...Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389/fams.2016.00002  ...and, uh, gentle reminder: keep your friendly neighborhood biostatistician happy by making your statistical...

...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted expression...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression...the same transgene: an optogenetic reporter, channelrhodopsin, fused to a fluorescent GFP marker (CatCH-...

...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...efficiently with SNAP-tag. If you want to use far-red rhodamine dyes for super-resolution imaging, HaloTag gives...

.... The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability ...separation of proteins of different sizes.  Table 2: Recommended gel percentages for various protein sizes...are consistent and stable at room temperature for 1–2 years and are available in formulas specific to your...response over a broad dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic...

...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...story (1). From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter...

... Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive inhibitory rhodopsin Ace2N and mScarlet...Ace-mScarlet, a fusion of a voltage-sensitive inhibitory rhodopsin (Ace2N) with the red fluorescent protein (mScarlet...green-fluorescent GEVIs and blue-light-sensitive channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids...

...by delivering an AAV construct expressing channelrhodopsin 2 (ChR2), a blue light sensitive cation channel... Plasmid tools for microbiome studies Channel rhodopsins for optogenetics Viral Vectors 101: Chemogenetics...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...Cas9’. The Cas9 nuclease is a genome engineering powerhouse, and for good reason. It is guided by an RNA ...

...also successfully regulated expression of channelrhodopsin-2, commonly used in optogenetics, opening up... Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “Cell type-specific manipulation...

...retroviral library was used to transduce T helper type 2 (Th2) cells from mice that constitutively express ...Gottschalk lab has surveyed a range of microbial rhodopsin GEVIs in excitable cells of the nematode Caenorhabditis...membranes, which might beneficially influence the rhodopsin properties. Find these voltage biosensors at Addgene...

... ligands can elicit varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic...fluorescent labels. In Nature Methods (Vol. 19, Issue 2, pp. 149–158). Nature Research. https://doi.org/10.1038...and mitophagy, can be tracked using dyes like Rhodamine 123 (R123) (Liu et al., 2017). In multi-color ...diffraction limit of light. Dyes like silicon-rhodamine or Janelia Fluor (JF) dyes have specific properties...

...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional...//doi.org/10.1186/1472-6750-6-43Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Cumate-inducible gene expression...

...light sensing proteins. Furthermore, during the first 2 weeks of life, larvae already exhibit distinct behaviors..."Optical control of zebrafish behavior with halorhodopsin." Proceedings of the National Academy of Sciences...