...construct, it exhibits modest levels of gene activation, with some genes experiencing around 2-fold activation.... This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou ...that requires modest gene activation. Synergistic Activation Mediator (SAM) Description SAM uses specially...-component system dependent on gRNA design (SAM) or peptide design (SunTag). This streamlines its delivery...genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9...VPR and SunTag) (Chavez et al., 2016). SunTag Description Rather than using a single copy of VP64 per each...expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex with dCas9 to ...

...required components are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...degradation of the tagged protein. Degrons are destabilizing domains which will be identified by various ...proteasome for degradation. Like several other tags described here, the dTAG uses a small molecule, a tag, and...protein for imaging. Given its popularity and widespread usage, a proteolysis-targeting chimera (PROTAC... The auxin-inducible degron (AID) system is the oldest system reviewed here. The AID system relies on ...Thankfully, this is a hot topic with many resources! In Bondeson et al, they review all the tags discussed here...

...PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA The gRNA should...Find the plasmids from this publication at Addgene. 2. Dalvai, Mathieu, et al. (2015). “A Scalable Genome-Editing...such a system is likely to introduce additional nucleotides flanking the insertion. If the DNA ends anneal...MMEJ may also introduce, substitute, or delete nucleotides in addition to the expected deletion, but the...Building on the lab’s previous work, Sakuma et al. describe a detailed protocol for MMEJ-mediated knock-in...binding in mammalian genomes as assessed by CRISPR design tools at crispr.mit.edu. They tested their system...

...amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria on...fluorescent proteins, it’s important to note that despite their distinction by name, chromoproteins are actually...chromoproteins have a shorter history. In 1987, scientists described an unusual blue-colored protein from the jellyfish... chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and ...recombination events: The plasmid carrying the desired genetic modification integrates into the genome...rhamnose-inducible promoter. The resolution step leads to the desired mutation and excision of the vector backbone. To...

...estimate transformation efficiency (Figure 2).    Figure 2: Example of a serial dilution to estimate...(Are the inserts of different sizes?), and the desired coverage for the library (How important is it that...  The most important factor to consider is your desired library coverage, since this will help decide what...similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can...should always experimentally test the protocol you design to ensure that it works for your library before...Addgene. If you have any specific questions about designing your library amplification protocol, feel free...

...Indicators GEVIs can be divided up structurally into 2 main groups: voltage sensitive domains and Rhodopsin...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically...fluorescent output. The GEVI family of gadgets includes Ace, JEDI, and many more. They can be targeted...component as well as a small molecule which we will describe in more detail below. Types of Voltage Indicators...Drosophila to phosphatases from sea squirts. This design structurally separates the two components of the...membrane, a concern with several of the previously described systems. Several adaptations of this method have...voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or both. Targeting these systems...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...tools, they do have undesired off-target editing effects. ABEs have been described to introduce base changes...efficient. Informed by these mutations and previously described TadA mutations (Gaudelli et al., 2017), they modified...heterodimeric fusion of wild-type TadA and TadA7.10, they designed one set of ABE8’s similarly (ABE8.x-d). In order...

...engineering methods have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’...Nature biotechnology (2016). PubMed PMID: 27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." ...researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene ...Try NgAgo for Yourself On the other hand, the widespread success of CRISPR-Cas9 across multiple model ...vector or in vitro synthesis, which could be less desirable if one has to target multiple sites in a single...of the supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly degraded by the cellular...

...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...results can be affected by DNA quality and primer design, particularly for amplicons under 100 bp (Ma, 2015... concern, look for the lowest-cost option that provides you confidence in the results you need.  Whichever...by running the product on a gel. And only ddPCR provides absolute quantitative data, instead of relative...Polymerase Chain Reaction (PCR) Protocol: How to Design a primer  Molecular Biology Reference Molecular...

... digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw cycles...PMID: 24003179. PubMed Central PMCID: PMC3760253. 2. Rohwer, Forest. "Global phage diversity." Cell 113.2... try amplifying the viral vector with bacteria designed to minimize rearrangements such as NEB Stable ...production of virus. Alternatively, if the virus encodes a fluorescent protein, the packaging cells could...working with, there’s a lot that goes into virus design, production, and transduction. Once you’ve harvested...

...from GFP and the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising..., V163A EBFP F64L, S65T, Y66H, Y145F   Table 2: Functional role of specific mutations in GFP derivatives...biochemical properties. In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence... in the wildtype protein and facilitating its widespread use in research. Many other mutations have since...array of GFP-based fluorescent biosensors has been designed to detect a variety of intracellular conditions...

... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody... and to work in a variety of applications. The desired specificity of your secondary antibody depends ...

...days before the deadline isn’t going to be much use. 2. Originality is essential for grant success The two... it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback from...to a lay audience. My lay paragraph typically includes a couple of sentences of introduction to my area...

... of permeabilization and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol ... at too high a concentration.      Figure 2: Examples of different permeabilization strategies...improve antibody binding for some targets, they can destroy it for others. That said, organic solvents’ impact...critical to the outcome of your IF experiment and deserve careful thought and attention.   * There are other...

...connectivity to other data sources used in research; (2) connectivity to other tools used in research; and...by making it easy to get data out of the ELN; and (2) facilitate inter-operability with other research ...reproducibility than others. Unfortunately, most ELNs are designed as closed ecosystems and act as data silos because...RSpace ELN supports reproducibility RSpace was designed in response to requirements that emerged from ...used in research, and data repositories. Below I describe how each of these kinds of connectivity facilitates... in institutional and lab servers specifically designed to hold large amounts of data. Although it may... use of other tools they use in research. This includes chat apps like Slack, Microsoft Teams, and Google...

...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 ...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)... large size of Cas9 activators, which largely precludes delivery by adeno-associated vectors (AAV). This...packaging capacity than AAV. Such an approach was described recently by the Day Lab, who developed neuron-...

...important role in solving the structure of DNA. 2. The Immortal Rap of Henrietta Lacks Students in Oakland...One of Tom's earliest and most popular videos. Describes fossil rocks and their importance in understanding...

... which one you choose depends on your experiment (de Ruijter et al., 2003). For example, we did our initial...GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate, which will then...Humana Press, 2009. 41-50. PubMed PMID: 19183892. De Ruijter, N. C. A., et al. "Evaluation and comparison...and a vacuum is applied with a vacuum bell or desiccator. Successful infiltration can often be seen by...

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve...individual molecules. Poor photon budgets are a widespread issue with fluorescent proteins—even the thriftiest...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical... the complexity of the system. A particularly desirable property for small-molecule labeling strategies...size, brightness, and cell permeability. In past decades, further refinements to this established dye structure...spectral range. Nevertheless, these fluorophores were designed as antibody and oligonucleotide labels for fixed-cell...

...et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade; although...cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT expression constructs. (B)...human therapeutics (e.g. using Sleeping Beauty). Despite their diverse utility, DNA transposons have been...(V. cholerae INTEGRATE - hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components...the same promoter, and a donor plasmid (pDonor) encodes the mini-transposon. pEffector and pDonor can also...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...even very large genetic cargo, at one or multiple desired genomic loci. Many applications of these systems...