...tools, to more scientific field-based solutions or open source solutions. In comparison to paper-based record...that, academic institutions tend to think about the long term, so they need a solution for which they ...organizing folders in complex ways, there are solutions that bring more to the table in terms of data ...understand the situation in depth and provide the solution that fits the most labs, our team conducted a ... once they establish the system within their institution. In the study, respondents were also asked about...comment reflected the desire for a flexible generic solution, rather than an ELN designed for a specific research... in the near future? As most payable software solutions have user-based pricing, it is good to know how...

...research tools: Sleeping beauty, PiggyBac, and Tol2.  Sleeping beauty Sleeping Beauty is a synthetic transposable...insertions can be detrimental, it also drives genomic evolution and is used as a tool by scientists to move DNA...fish (Sandoval-Villegas et al., 2021). Sleeping Beauty’s preferred target site for integration is TA dinucleotides... efficiency decreases with cargo size. Sleeping Beauty has a close-to-random integration profile in mammalian...transposition activity (Voigt et al., 2016). Sleeping Beauty plasmids  piggyBac Although its name suggests...and DNaseI hypersensitivity sites. Like Sleeping Beauty, piggyBac has an integration efficiency in human...caused albinism (Koga et al., 1996). Unlike Sleeping Beauty and piggyBac, Tol2 has a weak consensus sequence...

...-Assisted Continuous Evolution (PACE) and Phage-Assisted Non-Continuous Evolution (PANCE) selection systems...rate of dilution (passage from the infected to a fresh uninfected host cell culture). This dilution step ...overall dilution and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing...certain sites, or make only a subset of base substitutions.  The need for speed: base editing before Cas...These systems allow for rapid continuous protein evolution through many rounds of mutations, selection, and...followed by higher stringency PACE, resulted in the evolution of TadA variants with much higher base editing... engineer and optimize base editors? Targeted evolution of naturally occurring enzymes and functional ...

...cap: institutions of higher education nonprofit entities related to or affiliated with institutions of ...role at a prestigious institution: This criterion is consistently under scrutiny. USCIS wants you to prove... Trump signed a "Buy American, Hire American" Executive Order which sets broad policy intentions directing...exempt from the cap if the applicant works at institutions of higher education, a related or affiliated...of the work of those in your field. Original contributions to the field that have had a major impact. For...and your continued work is necessary to create therapeutics to cure the cancer. Authorship:  Publishing ...professors. Casseus Law has seen that research institutions are willing to sponsor postdoctoral researchers...

...developed to break through the resolution barrier while still utilizing fluorescence microscopy, is another...previously only visible via techniques like super-resolution microscopy or electron microscopy. Since its ...light microscopes are limited in the level of resolution that they can achieve. This means that regardless...Dani, et al., 2010). How do you overcome this resolution limit? Electron Microscopy (EM) is one option...specific targets you can label in a sample. Super-resolution microscopy (SRM), a family of imaging techniques...more accessible imaging equipment, ExM protocols utilize relatively inexpensive reagents and are readily... What are some of the limitations?  A “super-resolution” technique that uses common reagents, straightforward...

...three tabs.  Each of the three tabs - Contributions, Indel Distribution and Traces - provides particular details...analysis screen. ICE analysis details Contributions   The Contributions tab shows the inferred sequences present...the most 5’ cut site.     Indel Distribution In the Indel Distribution tab, you’ll find an Indel plot ... blog. CRISPR-based genome engineering has revolutionized the gene editing field by making experimental... useful measure to determine how many of the contributing indels are likely to result in a functional ... interested in understanding how many of the contributing indels are likely to result in a functional ...plot (left panel) which displays the inferred distribution of indel sizes in the entire edited population...

...diagnostic tools and therapeutics. In addition, the strategic use of whole phages as therapeutics, which is also...blocking solution (around 108 CFU/mL is a good place to start) and add your membranes to the solution, shaking...little about how they infect and influence the evolution of their bacterial prey. Currently, receptor binding...be understood. Therefore, in addition to their utility in the health sector, phages represent a reservoir...overnight at 30°C is a good start. Prepare a lysis solution containing your favourite bacterial protein extraction...thick filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When your...membranes off the plates and place each onto a lysis solution-soaked filter paper (colony-side up) and leave...

...targeted point mutations for directed evolution Directed evolution is an important method for protein engineering...specific agonists for orphan GPCRs demonstrating the utility of this platform for drug discovery. Researchers...Ras oncogenes have been difficult to target therapeutically due in no small part to the central role Ras...CCP); the latter of which greatly enhances the utility of this collection as it can be used to easily ...CRISPR library Addgene has had the privilege of distributing several human and murine CRISPR pooled libraries... allows for easy assembly of DNA fragments by utilizing regions of sequence homology (usually 30-40bp ... are available at Addgene. Since GMAP assembly utilizes 5 different overlap sites, this technique also...

...all-in-one acid-guanidinium-phenol solution that combines the homogenization solution and phenol addition of the...and less of being left with a bunch of random solutions when all the spin columns run out. In this article...suppliers and glassware should be treated with a DEPC solution for 1 hour, and autoclaved to remove residual ...homogenized spleens in a guanidinium thiocyanate solution and spun down the homogenate to remove the insoluble...homogenized and lysed in a guanidinium thiocyanate solution. However, instead of RNA separation using cesium-chloride...RNAProtect® from Qiagen, are ammonium sulfate-based solutions that work by inhibiting RNase activity within ...use of TRIzol® and ammonium sulfate stabilizing solutions can be made in house.   A common problem with ...

...temperature. If the agarose solution is too hot, the cells will die. If the solution is too cold, it will clump...out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time...survive at low densities - adding to the expense of dilution cloning. Taking a page from bacterial cloning...different densities for selection). A 2% agarose solution is then melted (and thereby sterilized) by microwaving...placed in the hood. Finally, a sterile 2X DMEM solution (diluted from 10X and containing both Hepes and... mm dishes only 1.5 ml of each are used). This solution is capped, swirled and tested for the correct ...the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body very sensitive to heat...

...happens, the solution can be cooled and the two phases mixed together. Lastly, the PEG solution needs to ... the iodixanol solutions and let me double-check that the iodixanol gradient solutions were prepared correctly...because it’s time consuming to prepare the 40% PEG solution. PEG takes several hours on a heated stir plate...to dissolve and requires monitoring so that the solution doesn’t get too hot because then the PEG will ...either by filtration, which takes time because the solution is viscous, or by autoclaving, which also takes...preparation. 15%, 25%, 40%, and 60% iodixanol solutions are carefully layered and then the viral suspension...titer for your AAV, remember to account for the dilution of the sample for the DNAseI digest. Use good ...

...repository has grown to include many CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative...cumulative numbers of CRISPR plasmid distribution and deposits (Figures 1A and 1B); the cumulative number...each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative CRISPR plasmids...Figure 2, the numbers on the X axis refer to distribution and deposits per year, rather than a cumulative...   Figure 2: Addgene CRISPR deposit and distribution by function and expression type. (A) Cumulative...4).    Figure 4: Addgene deposits and distribution by country. (A) Total CRISPR plasmids deposited...publication, “The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene...

...tasks, and then executing on them in a timely fashion while allowing for creative solutions to emerge. They... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance...clinical research to discover and test the best new therapeutics. Consultants often work with such teams to help...associated with the business. Commercial teams routinely work with consulting firms on projects such as...future, and yet are incredibly difficult to find solutions to. There are many reasons why client situations...at the core of the suite of benefits a given therapeutic approach brings to patients, doctors, and insurance...others go on to work with commercial teams at pharmaceutical companies, nonprofit or global health organizations...

...DNA prep. You might see protocols that recommend eluting in water, Tris-EDTA (TE), just Tris buffer, or ... main factors that can affect DNA stability in solution are pH and DNase contamination.  Extreme pH can...activity. For these reasons choosing the correct solution is fundamental, and it will impact the time you...which experiments you can use your plasmid.  DNA elution choices: TE, Tris buffer, or water Figure...two of the choices that companies offer for the elution of plasmid DNA through their kit. The advantage... plasmid in every type of experiment. The two solutions may differ though in their capacity to maintain... to store it and for how long. The most common solution is to keep your plasmid at -20°C or even at -80...

...to the distribution of the submitted material under the terms deemed necessary by my institution or other... pre-publication plasmids" and set a desired distribution status. You can submit "Published plasmids":...description of the deposit. Select your desired distribution status: Hold for publication: we will initiate...pending QC: your plasmids become available for distribution as soon as they pass our standard QC process...that will make it easier for scientists to best utilize your plasmids (these can be protocols, GenBank ...information for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should ...Survival strain. Figure 10: Enter the growth and distribution information. Step 5: Verification On this page...

...c-terminal nuclease domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer ...activation Optogenetics is a powerful tool that utilizes light to control and monitor individual living... both live cells and whole animals. The system utilizes the bulldozer-like quality of the ribosome during...red. This system achieves great specificity and resolution by using 6-12 phage PP7 coat protein binding ...create an Entry vector containing a promoter for constitutive (CMV, SV40, SFFV) or inducible (CMV-TO) cDNA...expression, sgRNA (U6) expression, shRNA/shRNA-miR30 constitutive (H1, U6, 7SK) or inducible shRNA-miR30 (CMV-...experimental purposes including (1) single or multiple, constitutive or inducible, gene knockdown with shRNA or shRNA-miR30...

...issues independently; for example, Harvard’s Therapeutics Graduate Program has a compulsory internship...It remains contested, however, whether a better solution would be to reduce the number of entrants into...in Boston - this time with the aim of finding solutions to some of the problems that were identified last...adequately supply these demands? Are there other solutions, such as increasing numbers in Master’s level ...the academic labour market. Her many notable contributions in this area include publishing the book ‘How...How Economics Shapes Science’ and contributing to the NIH’s Biomedical Research Workforce Working Group...2015 where we hope to begin developing concrete solutions to the problems looming over the academic research...

...2020). Directed evolution of adenine base editors with increased activity and therapeutic application. Nature...efficiency (Koblan et al., 2018). Later directed evolution efforts yielded an editor with more flexible sequence...DNA adenine deaminases. The team used directed evolution to create one from the RNA adenine deaminase TadA... TadA.   After seven rounds of molecular evolution, they obtained four adenine base editors (ABEs). ABE7.10...In this first, the Liu lab used phage-assisted evolution selection systems to generate ABE8e(TadA-8e V106W...alternative approach, engineering and directed evolution of the TadA domain has yielded base editors that...Tolar, J., Osborn, M. J., & Liu, D. R. (2023). Evolution of an adenine base editor into a small, efficient...

...2020) Directed evolution of adenine base editors with increased activity and therapeutic application. Nature...primary human cells. Now, scientists at Beam Therapeutics have found a way to improve editing in primary...Image from Gaudelli et al., 2020. Directed evolution identifies adenine base editors with improved ... and Head of Gene Editing Technology at Beam Therapeutics, used a previously developed bacterial selection...ABE8.x”: “8” stands for the eighth round of ABE evolution and the “x” is the placeholder for the number ...editing specificity, for example for potential therapeutic approaches, the authors recommend mRNA delivery...off-target deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved...

...proteins that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These...et al, 2017). To add insult to injury, the most utilized PAM sequence, that of SpCas9, is NGG. Assuming...Identifying PAM-free and -flexible Cas enzymes   The solution to the PAM problem seems simple, in theory: find...options, none of them offer a one size fits all solution to the issue. Nor are all Cas enzymes equal; just...generated by the Liu lab through phage-assisted evolution, specifically with the goal of generating Cas ...-less? Have we reached an essentially PAM-free solution? Almost! Assuming a PAM of NRN, 50% of randomly...History of CRISPR Cas – A Tale of Survival and Evolution  ...