...Klompe et al., 2019; Strecker et al., 2019), which direct RNA-guided transposition. By natively combining...I-F CRISPR-Cas system. The four major components required for RNA-guided DNA integration are (Fig. 1A, top...transposition in type I-F INTEGRATE. The requirement for multiple Cas proteins to form the Cascade...targeting (Halpin-Healy et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by...instead of three Cas proteins (Fig. 1A, bottom), and require only two transposase proteins (TnsB and C). While...effector plasmid (pEffector) expresses all seven required genes and the CRISPR RNA under the same promoter...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...

...fluorescent donor to your protein is to hardwire the molecule directly into the protein’s peptide sequence...(the donor) is excited, it can transfer energy directly via resonance to the second fluorophore (the acceptor...translation (Ermolenko et al., 2007). All that is required is that the proteins/nucleic acids/lipids of interest...delivered to the right place? FRET experiments require targeting the donor and acceptor fluorophores to...experimental design is that the targeting specificity requirements are different for the donor and acceptor fluorophores...codon (often amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural...overhaul of the cell’s native tRNA synthetases is required and thus far, this method has proven most effective...

... ori and requires the host proteins DnaA and IHF to initiate replication. But pSC101 requires a third ...RepA binds to directly repeated sequences called iterons located near the ori and directly interacts with...said to be under relaxed control, as they only require the host's replication machinery for elongation...have enough nutrients available to complete the entire round of replication. Initiation is the primary...versions, but binding of the low-affinity versions is required to start the replication process. When enough ...creates two replication forks that move in opposite directions away from the ori. In the elongation phase, a...on host-encoded proteins, it can be regulated indirectly via the same mechanisms that affect DnaA and ...

...is necessary to be direct and clear. There is place in human discourse for indirect language, but it is...Giving timely and useful feedback is an absolutely required aspect of being a successful manager. Great bosses...over, what would you do differently? Beware of indirect language You must be honest about performance ... should be aware that they are at risk of being fired and should be looking for a new position. One of... mistakes I see is managers who hint at or use indirect language to talk about real performance issues...issues. Watch this little video to learn more about indirect language from Steven Pinker. As a manager you ...Scientists. Subscribe to the Addgene Blog to follow the entire series as it is published. For more reading try...

...practices, their opinions towards ELNs, and their desired functionalities i.e. main priorities of different..., and the fact that long-term use is likely to require on-going maintenance and support.” Besides that...it as very important. One comment reflected the desire for a flexible generic solution, rather than an...switching from paper to an electronic lab notebook requires time and knowledge. In a recent article, we translated...reporting on a project – printing your data out can be required every once in a while, so it is good to make sure...your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering this... that take any ownership over your data or have direct access to it. Your data should be kept private ...

... for a site-directed mutagenesis PCR that uses mutagenic primers to introduce all desired codon changes...generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure...phage-derived uracil labeled template, this method requires further DNA shuffling to combine pairs of mutations...of your liking. This method is similar to site-directed mutagenesis (SDM) PCR, but with a twist since ...the plasmid. Again, if they are in the opposite direction, both strands of the plasmid will be nicked during...of your plasmid. If the sites are in opposite directions, you just need to use a plasmid with BbvCI restriction... the gene or use custom gene synthesis or site directed mutagenesis to remove the BbvCI site(s). Overall...

...but this approach would require robust stem cell editing to maintain the desired phenotype. Precision genome...skipping. Addgene depositor Charles Gersbach used paired zinc finger nucleases to remove exon 51 in DMD ...For such a therapy to be successful, multiple requirements must be met. First, CRISPR would need to be ...especially well suited for genome editing. The homology-directed repair (HDR) pathway is downregulated in mature...is maintained for at least six months. Future directions and obstacles to clinical CRISPR editing Given...NHEJ-mediated editing, but most other diseases will require HDR-mediated precision editing tailored to smaller...:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya...

...the requirement for digestive motility to not be disturbed, the brain fibers could not be directly repurposed...constantly influenced by signals it receives from the entire body, making it difficult to probe due to the lack...monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes of interest, and stimulate...blocks of polymers are heated up and pulled to a desired thickness. The fibers are drawn with microelectronics...the fiber horizon? The brain interfaces with the entire body, so the obvious next applications are to expand.... P. (2020). Modular Optoelectronic System for Wireless, Programmable Neuromodulation During Free Behavior... Multifunctional microelectronic fibers enable wireless modulation of gut and brain neural circuits. Nature...

...appropriate plasmids, and code the entire page. That would be entirely too much work for one person, it ...Clearly articulate the desired outcomes. Begin with the end in mind. Specify desired results. Are you delegating...and are experts in each of the individual tasks required to develop the web page. The team manager must... expert) the end result is high quality and the entire process is completed more efficiently. Listen to...criteria for delegating tasks appropriately: Are the desired goals and outcomes clear? If you don’t have clear...results. What does success look like? Agree on desired outcomes. Concern yourself with what is accomplished...

...CyOFP1-NLuc-CyOFP1; Antares). Direct injection of plasmids for Antares or firefly luciferase into mouse livers... like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase...brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase. This is important...for how sensitive you can make a reaction. NLuc requires 100-fold less protein to produce the same amount... wavelengths (Fluc emits at 610 nm) and do not require external light sources. Unfortunately, detecting...detecting traditional luciferases’ dim light emissions requires long acquisition times that may be impractical...changes in intracellular calcium and is useful for direct imaging of events in cells and brain explants. ...

...(2021). Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery...proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor ...Flannery, J. G., & Schaffer, D. V. (2013). In vivo-directed evolution of a new adeno-associated virus for ... lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid...Adeno-Associated Virus Serotype 4 (AAV4) and AAV5 Both Require Sialic Acid Binding for Hemagglutination and Efficient...Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections. Human Gene Therapy, 30(12), ....2019.127 Sands, M. S. (2011). AAV-mediated liver-directed gene therapy. Methods in Molecular Biology (Clifton...

...absolute measure of target abundance, does not require standard curves, and can theoretically detect a...what kind of assays can be used to sniff out your desired mutations. The assays you will use aren’t that ...either a donor sequence integrated through homology-directed repair (HDR) (1), or indel mutations resulting...non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation of NHEJ to generate... and a NHEJ/ “drop-off” probe designed to bind directly at the predicted double strand break site (Figure...quantification. Comparatively, mismatch assays require between 200 and 500 ng of purified PCR product.... those with the bi-allelic mutations typically desired when screening clones for complete functional knockout...

...are measured. While the entire genome can be knocked out with CRISPR, the entirety of the small molecule... another way, only cells or organisms with the desired phenotype will survive and be assayed at the end...phenotype in the surviving population. Selections do require there to be a culture condition that can apply ... have to be addressed with screens. Selections require there to be a culture condition which eliminates...then assay cells which survive as these must have acquired a favorable mutation. But if the cellular pathway... with cellular redundancies, a screen would be required to answer your experimental question. Now that....1246981 Leemhuis, H., Kelly, R.M. and Dijkhuizen, L. Directed evolution of enzymes: Library screening strategies...

...Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenence within an E. coli cell... across many different cell types and each one requires different elements for vector propogation. This...cerevisiae; however, we’ve also noted some features required for the replication of fission yeast, Schizosaccharomyces...These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination...for use in S. pombe, on the other hand, do not require a well defined ORI. Instead, the size and A-T content...organism to synthesize a particular organic compound required for its growth. Many auxotrophic strains of yeast...they have incorporated the plasmid carrying the required gene.  The table below lists some of the most...

... antibody was directed toward a protein of interest but the second antibody was directed toward AHA. Now...these experiments are problematic because they require radiolabeled amino acids. The use of radioactive...radioactive materials is highly regulated, requires designated work areas and come with health risks. Importantly...Erin M Schuman, a former Caltech researcher, now Director at the Max Planck Institute for Brain Research... In the standard PLA assay, primary antibodies directed towards two endogenous proteins of interests, ...Central PMCID: PMC300121. 3. Söderberg, Ola, et al. "Direct observation of individual endogenous protein complexes... PubMed PMID: 17072308.  4. Dieck, Tom, et al. "Direct visualization of newly synthesized target proteins...

...graduate students and postdocs will be hired, direct and indirect costs, etc. It may be helpful to ask ...Research at universities typically requires funding from a variety of government and academic institutions...skills, Elucidates the big picture and future directions of the project, Outlines a detailed scope of...! Additionally, keep in mind that universities require application submission in-house approximately 2... RFA contains all of the important information required for a successful grant application. It typically...avoidance of ‘to be’ verbs creates strong, well-directed meaning and delivers strength to the sentence....

...Nammour, J., Maiz, D., Dujardin, S., Ramirez, S. H., Hudry, E., & Maguire, C. A. (2019). Selection of an Efficient...(2021). Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery...off-target expression, it may be better to choose a direct delivery method over systemic delivery. Otherwise... packaging Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict...expression to specific tissues and cell types. If desired, spatiotemporal control of transgene expression...and astrocytes located in the CNS, however, it requires high doses. Requests have shifted toward the more...effectively transduce astrocytes or oligodendrocytes when paired with appropriate cell type-specific promoters or...

...extra level of safety, they also require a fourth plasmid. The requirement for additional DNA can impact ...third generation systems such that it no longer requires the viral transactivator, Tat. For more information...increasing the number of recombination events that are required to generate RCL. To overcome the titering hurdles...unconcentrated prep. With this in mind, if the experiment requires a certain volume of virus, consider scaling up...larger vessel, such as a 3 or 5-layer flask will require some initial optimization; cell number, media volume... method is relatively inexpensive and does not require an ultracentrifuge, PEG will precipitate all proteins... may not be the best method when pure virus is required downstream. When assessing lentiviral titer, don't...

...E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  a guide RNA...accessible. Image from Gaudelli et al., 2020. Directed evolution identifies adenine base editors with...editing in cell lines Nicole Gaudelli, the Associate Director and Head of Gene Editing Technology at Beam Therapeutics...insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a problem. Therefore...editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA to contain ...editors are precise molecular tools, they do have undesired off-target editing effects. ABEs have been described...off-target editing frequencies. For applications that require high DNA editing specificity, for example for potential...

...expand applications that require very specific targeting, like homology directed repair and base editing...order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence...David Liu’s lab, have gone one step further, using directed evolution to create xCas9, an enzyme recognizing...more complicated if you’d like to use homology directed repair to make precise edits or knock-ins. Editing...other species, like S. aureus and N. meningitidis require different PAM sites than SpCas9, in this case, ...broader PAM flexibility. Creating xCas9 through directed evolution Hu et al. used phage-assisted evolution...about CRISPR Base Editing Learn about Homology Directed Repair Resources on Addgene.org Check out our...