...Image courtesy of Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP ...functions through macropinocytosis. D’Astolfo et al. tested iTOP in a series of primary cell types using Cre...Cas9 is all the rage, they had another reason to test this system. It’s difficult to determine how much....2015.03.028. PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous Protein Delivery...

...that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm (Table 1), which is near...line, Figure 2). The yellow/green laser, however, excites at 561 nm, which is close to the global maximum...more photons are absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   ...the same laser type. Knowing that the red laser excites at 640 nm (Table 1), while comparing the two fluorophore's...is about 750 nm. If we were to use a laser that excites at 750 nm, APC-Cy7 would show a much brighter signal...absorbed and emitted by matter, and how this process creates visible colors. Next, you learned about how fluorophores...

...of pure product in an affordable way, researchers test many different combinations of promoters, terminators...Protein expression in yeast MoClo is also for eukaryotes! The technology has been used to optimize protein... chromosomal integration of larger expression cassettes (Shaw et al., 2023). Those looking to express ...cutting. Beyond sgRNAs, MoClo can also be used to test a variety of Cas enzymes with different PAM requirements...applications (gene insert = Cas9 + gRNA), Addgene distributes specialized CRISPR kits too. The AspFlex Kit ...delivery. While Fragmid developers were specifically testing CRISPR tools, it should be easy enough for researchers...

...on a protein, enabling the labeling of protein substrates/peptides and antibodies. Example applications...intensity at ~85% of its original value after 60 minutes of illumination, highlighting its reliability for...G-protein-coupled receptors (GPCRs). Mutations in receptor sites can affect the fluorescence emission of labeled ...assay results (Hayashi-Takanaka et al., 2014)​​. Conjugates with lower DOL tend to have weaker fluorescence...., 2016). When selecting dyes, it's important to test them under experimental conditions. Common issues...toggle’ between fluorescent and non-fluorescent states in response to a specific stimuli, which leaves...

...Producing yeast expression cassettes Traditionally, generating expression cassettes for yeast transformation...in the amplification. This tool allows instant updates as fragments are added, deleted, or reordered and...Cloning sgRNAs Production of Yeast Expression Cassettes Generating constructs with multiple point mutations...typical two oligos, is more cost effective and eliminates the annealing step usually used to create a double-stranded...

...sessions and reported the intel back to my shyer classmates. For my undergraduate degree, I majored in human...stigma around sexual and reproductive health issues creates conditions under which misinformation can flourish...anxious, whispered questions from friends and classmates back in high school, and it’s oddly fitting that...to read short abstracts comparing the mortality rates between two controversial health issues. One group... the National Academy of Sciences of the United States of America, 114(36), 9587-9592. doi:10.1073/pnas...

...diseases, and several treatments are currently being tested in clinical trials. It is speeding the process ...cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA targeting the...CFTR) by homologous recombination in cultured intestinal stem cells of patients with cystic fibrosis (...also really need to get a handle on how cleavage sites are repaired.” Those insights will be critical for... "Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients...

...sequence and ultimately its degradation. While other eukaryotes don’t naturally have this response, they do contain...protospacer adjacent motif) in order to bind the target sites for editing. PAM sequences can be from 2-6 base ...which mediated a C to T conversion in the target sites adjacent to an NG PAM in human cells. The increased...and its variants can be used to engineer specific sites that were previously difficult. Find SpCas9 and...the Tara Oceans Project datasets. This study demonstrates the power of combining the analysis of publicly...

...TCSs). TCSs consist of a histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses...far-red light or dark conditions, the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription...below). Subsequent exposure to red light rapidly deactivates the system. Voigt’s lab used this system to develop...far-red light or dark conditions, the chromophore activates the response regulator via phosphorylation, promoting...reporter. Subsequent exposure to red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive...

...your resume? How can you stand out among all the candidates applying for the position?   Here are a few things...people choose a reversed chronological style (the latest to the earliest) or a functional one. Pick one ...surprised to learn how often I see resumes from good candidates that I almost missed because I had to “dig” hard...topic will give you some advantage over other candidates. Write about your transferable skills Some of...career services offices. You might think only undergraduates use these services, but some of the general...

...perfect. They may be slow, can only target certain sites, or make only a subset of base substitutions.  ...better base editors with the help of phages! The latest ABE from the Liu lab is ABE7.10 which contains ...the phage is not infectious. The editing speed dictates which phage variants can propagate faster than...compared to the original TadA-7.10 enzyme. After testing numerous TadA variants, they identified TadA-8e...all Cas9 and Cas12 homologs that the researchers tested, including SpCas9, SaCas9, LbCas12a, enAsCas12a...

...rather than a perfect lesson plan or outstanding test scores.  As a result, Kuldell launched the BioBuilder... fields rely on rapid prototyping to develop and test hypotheses. Students then use cellular extracts ...means to rapidly prototype DNA circuits and logic gates. This BioBuilder-Caltech collaboration has resulted...classroom poster — with both of these resources road tested over the summer with dozens of teachers around ...approaches and scientific know-how to design/build/test their own project ideas using synthetic biology....

...randomly, while others use attP sequence docking sites so that you can control exactly in which locus your...different loci. Fortunately, there are many docking sites available for integration. 2. How soon would you... development, organs of interest, such as the intestines, can be dissected at various times throughout... resources of the Drosophila community to easily test your hypotheses. Many thanks to our guest blogger... H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila...

...after this initial launch so stay tuned for more updates. Overview of SnapGene powered plasmid maps at...be detected by our in-house software and also eliminates many of the extraneous ORFs that cluttered our...identified a plasmid that you want to learn more about, updates to our sequence analyzer will allow you to dive...above and their functions. We hope that these updates will make it much easier to browse the repository...improvements. Let us know what you think about the updates in the comments section below and keep checking...

...interactions. It also can’t recognize PAMs, so it associates with random DNA sequences (Sternberg et al., ...sequencines. This helps explain why Cas9 can’t locate PAM sites until after RNA loading: the structure to interrogate...for complementarity with its guide RNA. But how to test DNA bases that are already snugly paired to each.... As mentioned above, however, Cas9 sometimes tolerates mismatches in the next 8-10 bases after perfect...conformations. This allows it to unwind the DNA and test for sequence complementarity with its guide RNA....California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber...

... it is standard practice to compare degradation rates between samples treated with or without an autophagy...ATG) in yeast. Atg8p (“p” stands for protein) associates with autophagosomal membranes, so tagging it ...monitor autophagy, but under some conditions it aggregates in an autophagy-independent manner. Additionally...induce autophagy, red but not green fluorescence accumulates in the vacuole. See Figure 2 for an example of...Autophagy. Methods in Enzymology Autophagy: Lower Eukaryotes and Non-Mammalian Systems, Part A, 33-42. doi...

...Monitor neo-substrates Small molecule degraders may recruit CRBN to nonstandard or “neo-substrates.” We advise...interest prior to starting knock-in experiments. Test dTAG molecule toxicity in new cell lines Treat parental...evaluation of degradation of known cereblon neo-substrates (IKZF1, IKZF3, GSPT1, ZFP91, and CK1a). While...While expression of these cereblon neo-substrates varies in different contexts (for example, IZKF1 and IZKF3...

...catalyzes the desulfuration of 3-mercaptopyruvate and generates an enzyme-bound hydropersulfide that is then transferred...biosensors or biosensors that detect new redox metabolites are vital for researchers to better understand...mechanical allodynia, a condition where a light touch creates a painful sensation. The dorsal horn of the spinal...perceiving touch inputs. In this system, light activates the neurons (via oChIEF) and a chemogenetic agonist...identify the mutants for further enrichment. Tian notes that this platform can be used to engineer many ...

...characterized by misfolding of the TTR protein, which contributes to the disease phenotype. Intellia initiated ...Biosciences is currently running this trial, with more updates expected in the coming months/years. The CRISPR-HIV... (Hussein et al). It employs Cas9 to cut at two sites within the viral genome to excise a large portion...disease, inflammatory diseases, blindness, and diabetes. Directions and obstacles for future CRISPR therapies... "Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients...

...A4 and A8-A10. The researchers selected 8 of the tested ABE8 constructs (ABE8.8-m/d, ABE8.13-m/d, ABE8.17...2 guanines). This limits the use of ABEs as DNA sites that do not contain a suitable PAM cannot be edited...for primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... gRNA used (Gaudelli et al., 2017). The authors tested ABE8.17-m and found that one base change mutation...overall improved base editing capacity, even at sites that used to be difficult to target. In particular...