...is would change over the next twenty years. But learn we did! From Addgene's very beginning, we had to...information about images that a sighted reader would glean from the image itself.  As we incorporated alt text...to alt text. We also were aware of the need for clear, accessible writing in scientific writing, and how...development workshop, “Make a Greater Impact Using Clear and Accessible Scientific Writing.” In this post... of writing: alt text (pun intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember...student using viral vectors for the first time and learning how to purify AAV.  Second, you’ll identify your...free to share your alt text in the comments! We learned a lot from listening to others' alt text descriptions...

...MEDLINE-indexed journal Nuclear Receptor Signaling. Dr. McKenna edits the website of the Nuclear Receptor Signaling... elicit their actions on other tissues. At the Nuclear Receptor Signaling Atlas (NURSA), one of our primary...regulated at the cellular, tissue and organ level by nuclear receptors (NRs), their coregulators, and their ...their physiological endocrine ligands. Nuclear receptors and their ligands NRs represent the largest family...Consortium represents a unique funding model in nuclear receptor research, and has generated a set of comprehensive...address many important and fundamental questions in nuclear receptor and coregulator research. Our Q-PCR datasets...for example, document the expression patterns of nuclear receptors across multiple different tissues in ...

...continuing the project after leaving the meeting. Follow the tips below to learn how you can run a top-notch...direction you’d like to pursue? Undefined scope can lead to long meetings spent on details that do not yet...our all-company meetings short, informative, and pleasant. Email: Sometimes, the right meeting is no meeting...screen using a Google Doc. Action items: Before leaving the meeting, skim back through your notes and pick... tend to get stuck diving into details, ask a colleague to help keep you accountable for moving along....your organization. Ask for feedback from your colleagues. When you attend a well-run meeting, note what...what worked well and ask the meeting leader for some tips. Before long, you will be running collaborative...

... Connie Cepko to learn about her management style. Leaders look to the future Leaders must get new things... they lead. Effective leaders rarely give orders (although they must occasionally). Good leaders exert...urged you to work on developing your “leadership skills”. Leadership is prized at all levels of an organization...figure out what skills were really leader-specific. How is leadership different from good management? Aren't...Manager, Leader” by Charlotte Beers, divides workers into three cycles: Doers, Managers and Leaders. I like... Mask: How Leaders Spark and Sustain Change, by Peter Fuda and Richard Badham Leadership Insights for...Business Review 10 Must Reads: On Leadership (Articles on Leadership, also can be purchased individually...

...wheel. Test different leaf ages Usually younger leaves work better. Not every leaf is equally susceptible...transient leaf transformation, the engineered Agrobacterium strain is brought into contact with the leaf tissue...Agrobacterium infiltration into leaves does not work well, as the leaves have a waxy texture and are difficult...transgenic plant can take even longer, a transient plant leaf transformation process could save the plant biologist...studied in hairy roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development...development of transgenic hairy roots takes at least six months. Generating a transgenic plant takes even...biosynthetic pathway. Why plant scientists do transient leaf transformations The development of transgenic plants...

...opportunities to learn from other biotech companies at workshops and can use what we learn to improve processes... end repair Adapter Ligation Clean up/Size selection Amplification Clean up  I found this kit easy to... post, I’ll tell you a little bit about what I learned from a New England Biolabs (NEB) hosted workshop...CosMCPrep system for this, but thought we could learn a little bit more about high throughput DNA prepping...class at NEB’s new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space...emerging biotech companies. The space was modern with clean bold colors of orange and white. The decor was very... this is the most important part of this newly released prep kit. In their prior NEBNext Ultra II DNA ...

... led for 5 years.  Different leadership styles Being an effective leader requires you to understand how...would come necessary changes. I also learnt to adapt my leadership style according to the needs of my committee...strengths and work effectively. Leadership responsibilities Being a leader in an organization, you need ...that come with leadership. The NPA was a great organization to develop these leadership skills in particular... These “leadership institute’ weekends would entail intense training to develop our leadership and management...sharing with other more junior leaders what I had experienced and learnt. Opening doors to further opportunities...develop my leadership and advocacy skills but will also put me in closer connection with other leaders in my...

...Considerations when choosing your Cas nuclease When choosing the Cas9 nuclease for your experiments, there are...but since then, scientists have adapted CRISPR nucleases for many more specific research needs. In this...determined by the PAM sequence recognized by the nuclease. For the most frequently used Cas9, spCas9 from...off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect match to the guide...difficult to determine, the potential for off-target cleavage should be an important consideration during any... measure of the on-target activity of the Cas9 nuclease. This can be determined by measuring the indel...activity divided by the off-target activity. Cas9 nucleases that exhibit enhanced site accessibility In order...

...neurons (measured using a red calcium sensor) leads to the release of serotonin (measured using the newly developed...shape and metabolism of leaves can be changed by light and temperature and leaf autofluorescence can interfere...explains. Developing an artificial leaf replica means creating a “leaf” with similar permeability of water...for bacteria. To compare the artificial leaf to A. thaliana leaf, Bernach plans to use a suite of fluorescent... determine whether the artificial leaf behaves like the living leave in this aspect. To monitor bacterial... the variant is unstable, it will be degraded, leading to reduced eGFP fluorescence. Cells expressing ...dosages.  Michal Bernach: Developing an artificial leaf replica system to study plant-bacteria and bacteria-bacteria...

...this tour of Addgene’s new conference rooms and learned some fun facts about these genes and the origins...

...://doi.org/10.1089/hum.2019.277 Lavin TK, Jin L, Lea NE, Wickersham IR (2020) Monosynaptic Tracing Success...BioRender.com   Causes of recombination and leaky expression While leaky expression and spontaneous recombination...will have some level of recombination that can lead to leaky expression in cells that do not express Cre ...scrambled cre-recombinase sites lead to greater frequency of reversion and leaky expression. On the right non-homologous...recombination between these sites may be the cause of leaky expression. To test their hypothesis, they randomly...recombination. This indicates that the amount of leaky expression can largely be attributed to the level... non-homologous scrambled recombination sights lead to reduced reversion and less off-target expression...

...RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in ...orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain...based system, require a single-component nuclease to mediate cleavage rather than the multi-subunit complex...contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease domain, indicating ...functional CRISPR nuclease that could be adapted for genome editing. The use of a different nuclease could potentially...that display robust cleavage activity similar to that of Cas9. These two nucleases, AsCpf1 and LbCpf1 ...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...

...Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015... crRNA to cleave target DNA (Cpf1 requires only a crRNA for target recognition and cleavage). C2c2 (now...domains to cleave target DNA. The HEPN domains within Cas13a are essential for RNA cleavage, consistent...bind and cleave RNA non-specifically. The ability of dCas13a to bind target RNA without cleaving the molecule...between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure... residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a), that is capable of binding...binding target RNA but lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR...

...gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied fluorescently...fluorophore that is quenched when intact and released when cleaved. Cleavage results in fluorescence and this approach... This activation results in a trans cleavage reaction that cleaves a nearby RNA sequence that contains...activated and performs non-specific collateral cleavage. This cleavage is coupled to fluorescence visualization...fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence is no longer quenched and can be ... a PAM (Protospacer Adjacent Motif) for target cleavage. This test can be performed in two steps for challenging...ssDNA-FQ reporters that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA...

...) to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How ...ssRNA cleavage byproducts. Thus, binding of Cas13 to the programmed region of interest would lead to both... the Addgene Blog Learn about using toehold switches to detect Zika virus   Learn more about targeting...degradation to develop the method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen...Both SHERLOCK and DETECTR harness the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA...ssDNA by Cas13 and Cas12a, respectively, to cleave and activate a reporter. The detectable signal from this...to the programmed sequence, it can promiscuously cleave surrounding ssRNA molecules. In SHERLOCK a quenched...

...for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage, but they...the two nuclease domains, Cas9 ensures both are positioned correctly before HNH or RuvC can cleave DNA, ...? It’s not actually clear! In vitro, Cas9 holds tightly to DNA even after cleaving it, so many researchers...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology...   Cas9 has two major lobes, called NUC (for nuclease) and REC (for recognition). The NUC lobe contains... and RuvC domains, which are named for similar nuclease folds found in other proteins. The C-terminal ... and helps recognize the guide RNA. Unlike the cleavage domains, the recognition domains—including the...

... strands hybridize, the nuclease can detect strands with mismatches and cleave them. The results are then...surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases." G3: Genes| Genomes...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015)...genotyping. The most common options include mismatch cleavage assays, such as Surveyor™, T7E1, and Sanger sequencing...adequate standards for validating edits.  Mismatch cleavage assays rely on pairing between the edited strand... in the genome predicted to contain off-target cleavage events. Unbiased techniques search the whole genome...

...sequence! In bacteria, Cas13d-mediated cleavage promotes collateral cleavage of other RNAs. As with other Cas13s...and two nuclear long non-coding RNAs. One interesting twist to Cas13d enzymes is their cleavage pattern...EsCas13d favors cleavage at uracils, but a more detailed exploration of this cleavage pattern is necessary...a CRISPR repeat array near a putative effector nuclease. The Cas13d proteins they identified have little...identified Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily...their own CRISPR arrays into guide RNAs. crRNA cleavage is retained in dCas13d and is thus HEPN-independent...Cas13s, this collateral cleavage does not occur when Cas13d is expressed in a mammalian system. Since Cas13d...

...and structural integrity; cleavable. Not great for purification; too cleavable in eukaryotes TAP GRRIPGLINP...FLAG) are cleaved by specific proteases without requiring the addition of an independent cleavage recognition...cell. FLAG tags can be cleaved by enterokinase, which recognizes DDDDK^X, cleaving after the lysine. The...Notes TEV ENLYFQS Cleaves between the Gln and Ser residues Thrombin LVPRGS Cleaves between Arg and Gly...maltose-binding protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful...sequence of amino acids, is basically a molecular leash for your protein. If you’re working with an uncharacterized...-based purification; has inherent enterokinase cleavage site GST Large Protein 26 Purification and Stability...

...characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern...detectable off-target cleavage. Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...Cpf1-mediated cleavage, the continued presence of the PAM may give Cpf1 the ability to cleave again and possibly...the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1 displays ...blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide...18-23 bases away from the PAM. Cpf1’s staggered cleavage pattern opens up the possibility of directional...