...by the Sternberg lab derives from Vibrio cholerae (V. cholerae INTEGRATE - hereafter VchINT) and encodes...and technical considerations Making insertions in bacterial genomes with the V. cholera INTEGRATE system... using Tn5), and as knock-in tools for human therapeutics (e.g. using Sleeping Beauty). Despite their ... CRISPR-Cas, these systems represent the next generation of tools for efficient and programmable insertions...achieved with VchINT, at two different incubation temperatures. Data adapted from Vo et al., 2020. In E...-recombineering, CRISPRa/i, and base editing. Leveraging a unique marriage between distinct biological...

... and several others can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical...by Roger Tsien, who showed that the selective interaction between a bisarsenical dye ligand (e.g., FlAsH... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent...corollary to these innovative labeling strategies, several groups—including mine—have been revisiting the ...PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve fluorophores for live-cell and...Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids from...

...After several decades of trying to address these challenges, researchers have amassed a considerable molecular... overexpress a recombinant protein? It is not generally in a cell’s best interest to overexpress a protein...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...for affinity and purification An affinity tag, generally a relatively small sequence of amino acids, is...Purification Binding and elution steps use very moderate buffer conditions FLAG DYKDDDD or DYKDDDDK or...interest would eventually get ridiculous (you generally don’t want more tag than protein), 2-3 tags is...IgG-binding domains. TAP has since come to encompass several other tag combinations, though frequently those...

...lines in tissue cultures takes half a year, and generating a transgenic plant can take even longer, a transient...are investigating how the production of cancer therapeutic drugs, vinblastine and vincristine, are regulated...transgenic hairy roots takes at least six months. Generating a transgenic plant takes even longer, is more... that neither a stable cell line or plant is regenerated from the transformed tissue. Gene function is...by working with young seedlings and optimizing several other aspects of the transient transformation procedure...Once we were on the right track, we switched to luciferases to more easily quantify the effect of the different...GUS::tMAS to confirm transformation success. Luciferases are great quantitative reporters and use a substrate...

...composed of actin and myosin which interact with each other to generate forces that cause cells and muscles...signalling pathway, which is a key regulator of all bilateral animal development. SHH plays a key role in proper...into the right and left hemisphere. Addgene has several plasmids that contain SHH, you can find them all...that are necessary for myosin activity and thus interaction with actin in nonmuscle systems in the fly. Spaghetti...other hand, is important in the formation of the visceral mesoderm which differentiates into the muscles...Embryos that lack bagpipe have severely disrupted visceral mesoderm and subsequently the gut does not form...is required for specification of the heart and visceral muscles in Drosophila." Development118.3 (1993...

...directly into at least one and preferably more than one of these five general purpose repositories. RSpace...own pros and cons. All ELNs have the virtue of liberating data from paper into an electronic environment...data out of the ELN; and (2) facilitate inter-operability with other research tools and infrastructure ...of Edinburgh, the University of Goettingen, and several other large research institutions. The following...repositories Over the past decade or so a number of general purpose repositories have been launched and are... associated with publications into one of five general purpose open source data repositories: Dataverse...one foot on the other side of the pond through interacting with colleagues in Edinburgh. You can follow ...

...workflows considerably easier, faster, and more efficient than previous methods. Still, generating reliable...sequence you used. The ICE tool will calculate overall editing efficiency and determine the profiles of...detect or analyze complex edits, such as those generated by multiplexed delivery of multiple sgRNAs. Since...particular parameter was automatically adjusted to generate ICE results. If there are no results or there ... the ICE linear regression is computed during generation of the ICE Score, the Pearson correlation coefficient...around the cut site. It shows, base-by-base, the average amount of signal that disagrees with the reference...sequencing bases downstream of the cut. Summary ICE generates NGS-quality CRISPR editing analysis from Sanger...

...what online tools people generally like to use to share ideas – with the general goal of seeing if and how...their disciplines. Included in the article is an interactive chart where you can see the yearly interdisciplinarity...abstracts identified. The journal Cell had the largest average view count per video abstract at 2,947 views. The...Gastroenterology (1,046 views) rounded out the top four average video abstract view counts.” For researchers looking...offers a low-barrier opportunity for researchers to leverage the video medium to communicate their research...their contribution. It offers a 5-step process and several examples for creating effective research videos... botanist and oceanographer who has a blog and several videos on sharing your research through video. ...

...detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence... to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How does...sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-... for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly...diagnostics (e.g. pathogen or virus detection), therapeutics and sensitive genotyping. The beauty of SHERLOCK...Cas12a however recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The... DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published this in Nature...

... are several factors that may prevent your gene from being up-regulated. For instance, generally, the ...known functions has the potential to be used in therapeutics, as it can increase gene expression at certain...be used to map molecular pathways and genetic interactions. Additionally, large scale CRISPR-based screens...Cas9 cutting activity, off target effects are generally not regarded as being a large problem for Cas9...driving aberrant transcription. That being said, we generally pick guides by putting the promoter of the gene...innovative technologies. To date his group has develop several popular CRISPR-based tools for altering DNA sequence..., Garnett MJ (2019) Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568...

...them remain uncharacterized. This represents a considerable barrier to furthering research and developing...Cancer Center tackled this problem by developing a moderate throughput functional genomic platform to annotate...over 1,000 mutations, and the group has deposited several hundred plasmids from this study with Addgene. ...research team identified 1,049 perturbations in several genes spanning 33 cancer types. The types of mutations...indels (Ng et al., 2018). From this data, they generated lentiviral constructs using the High-Throughput...cell lines and any mutations that that confer proliferation advantages will be revealed in the absence of...is important for the development of targeted therapeutic interventions for cancer. This screening platform...

... gene expression in yeast Megan McClean’s lab generated an optogenetic toolkit that controls repression...frequency of genome-wide off-target DNA editing. Using several rapid and cost-effective screening methods, David...with reduced Cas9-independent deamination. They generated these editors using the BE4max architecture. The...reported in this study have ~10-100-fold lower average Cas9-independent off-target DNA editing, ~5–50-...off-target DNA editing, and maintain ~50–90% of average on-target DNA-editing levels relative to BE4max...division, and cellular differentiation, but it takes several weeks to delete or deplete individual genes. Michael...depending on transgene design and delivery. They generated hPSC lines with AAVS1-integrated, inducible, and...

... visualize their activity. In 2019, the first generation of norepinephrine sensors was developed, named...norepinephrine (Feng et al., 2019). But this first generation of tools had limited sensitivity and speed and... models of nLightG (left) and nLightR (right) generated using ColabFold (Mirdita et al., 2022). B, Representative...Speed One of the major advantages of this next generation of norepinephrine sensors is their speed. nLightG...GRABNE were more sensitive than nLightR, both generating a measurable signal after electrical stimulation...test point, ten pulses. This is in line with the general findings that red fluorescent proteins tend to ...improvements. In particular, they’re focused on generating variants with improved affinity and dynamic range...

...process, I’ve provided guidance and mentoring to several people that found my insights helpful. All went...of candidates either get no interviews, or get several interviews but no job offers, is a failure to clearly...understand your proposal to give you proper consideration. 3. Present an easy to follow seminar See item...techniques. You DO need to be prepared to know the literature of your field and all of the caveats and alternatives...human health. Failure to do so is implicated in several human diseases. Proper patterning of cells and ...that we can manipulate to study pathology and therapies." This is only the first few minutes of the...incorporated example documents. This part has generated a lot of positive feedback. Many of these documents...

...believed that the exonuclease activity of the polymerase generates sticky ends in the PCR reactions. These ...amplification of a recombinant DNA without sequence alteration. Molecular cloning techniques are some of the...its advantages, most scientists still encounter several struggles with these techniques: unwanted mutations... excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific sequences for base...any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations introduced into...efficiency is highest when using high fidelity DNA polymerases like phusion. An 18-cycle PCR leads to the most...

...language is ... The general representation of data is... The figures are ... In general, I think that this...the study to be replicated or omission from the literature. I think that delivering constructive critique...studying plant development and abiotic stress tolerance, I have been peer-reviewing journal submissions... detailed. I prefer to do it on another day - preferably the following morning - as usually deadlines ...language in the first (and clunky) version of my general comments and adjust them if my opinion changed ..., increasing the impact of the work, and as a collateral benefit, increasing the impact of the specific...

...thoroughly answered by experts in human-computer interaction (HCI), and we have adhered rigorously to HCI ...qualities is an ongoing process that involves iterative refinements in response to customer feedback. ..., and then crafted a detection algorithm that tolerates occasional mismatches or indels. Empirical tests...effort to be rigorous, we dug into the original literature, some of it decades old, to provide reliable ...to the database. Another limitation is that by tolerating mismatches, our algorithm could annotate a feature...to newly sequenced strains through our mismatch tolerant detection algorithm and thereby speed up the annotation...the idea stage through all of its post-launch iterations.     Additional Resources on the Addgene Blog...

...address this, several years ago we partnered with Seqwell to establish a next-generation sequencing (NGS... by Single-stranded Virus Next-generation Sequencing. Molecular Therapy - Nucleic Acids 4:e260 . https...of the VGS process and use cases in Human Gene Therapy. Existing methods for AAV quality control Historically...Viral Genomes in Academic Settings. Human Gene Therapy Methods 30:195–205 . https://doi.org/10.1089/hgtb...Consistency Evaluation of AAV Preparations. Molecular Therapy 25:1375–1386 . https://doi.org/10.1016/j.ymthe.2017.04.001...

...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should...worry less about sequence optimality because you generally have fewer sequences to choose from). Finally,...non-homologous end joining (NHEJ) often leads to the generation of indels and thus frameshifts that disrupt the... the introduction of a specific mutation, one generally relies on homology directed repair (HDR) to incorporate...user may need to optimize numerous parameters to generate the desired edit. The wrench: Gene activation ...

...of proteins, and can be a readout for protein interaction studies such as immunoprecipitation and pull-... be assessed from a single western.  There are several variations of the protein transfer step including...wet, semi-dry, and dry but all follow the same general principle where the gel and membrane are “sandwiched... antibody should you use? You will likely find several different antibody options targeting your protein...example, in immunoprecipitation (IP) the antibody interacts with a protein in its native state while a western...are typically conjugated to an enzyme, such as horseradish peroxidase (HRP), that emits light upon reacting... cellular assays. They can determine protein interactions following immunoprecipitation and are a common...