...patients have achieved persistent immunological reconstitution with a normally functioning T cell repertoire...variety of vector delivery technologies have been investigated including lentiviruses, adenovirus, and inorganic...? There are a variety of key properties that distinguish AAVs as gene delivery tools: Unlike most naturally...repair via homology directed repair (HDR). This characteristic makes AAV vectors especially amenable to correcting...but not limited to hemophilia B, LPL deficiency, Cystic fibrosis, Muscular Dystrophy, Parkinson’s disease...overcome key challenges to their efficacy:  Pre-existing immunity: Many individuals already have wt AAV...high as 70% among the human population. These pre-existing antibodies can interfere with virus-cell interactions...

...technological advances from exacerbating existing inequalities and injustices. The Personal Genetics Education...organizations, libraries, youth groups, and faith institutions. The conversations often move quickly into sensitive...sensitive topics and uncomfortable places, from sticky ethical dilemmas to dark episodes in the history...hands on deck” to take care of the organizational logistics. Setting yourself on the path to science policy...being very focused on narrowly-defined research questions in our field of interest. We often communicate... on public lectures, science cafés or science festivals, and getting experience in either giving or running...

...CrispyCrunch may evolve over time, you can depend on it to stick to a few guiding principles that we've followed ...can see all the current code on GitHub, ask us questions about it, or send us a pull request for improving...genome browser, Ensembl, and others. It builds on existing popular web services, currently Crispor and Crispresso...designed outside CrispyCrunch, or you can input pre-existing guides for primer design. The ecosystem of CRISPR...deciding to build our own tool, we looked hard at existing tools. As has been written here on Addgene, there...HDR. Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide...hope it will do the same for you. Send us your questions or comments to [email protected]. We'...

...wealth of potential, and while many applications are still yet to be demonstrated (or invented), its behavior... into a variety of optogenetic tools, they’re optimistic that MagLOV can be similarly adaptable into future...-publishing directly on his website (and often posting updates on social media) and freely sharing code...results early and often, including works-in-progress still in need of further study and development like MagLOV...MagLOV. The results with MagLOV raise broader questions like “How and why do fluorescent proteins respond... speculate, they know they can’t answer these questions alone. They readily admit they don’t fully understand...more. If you’re interested in working on these questions or collaborating, the York Lab is eager to hear...

...antibody come into play? We’ll dive into those questions in this post as we explore… the life and times...and monoclonal antibodies. However, it was (and still is) infeasible to generate highly specific antibodies...target and basically impossible if you wanted to distinguish between endogenous and exogenous versions of ...significantly between human and the other species, suggesting that these antibodies recognize an epitope within...suitable pair for protein tagging. In two studies investigating mechanisms of endoplasmic reticulum localization...EQKLISEEDL) - larger than the substance P peptide, but still quite small. However, unlike the substance P tag...involve taking the Myc1-9E10 variable regions and sticking them onto different scaffolds. But over the years...

...Twitter - Tweet suggestions and tips with #NgAgo The Addgene Blog - Leave suggestions and tips in the ...November 18, 2016): Researchers from a variety of institutions recently reported their inability to recapitulate...nearly 400 times. Researchers have been excitedly testing NgAgo's genome editing applications. So, how does...successful, and the second section asked basic questions about the NgAgo protocols used by respondents ...epitope knockin was achieved by a few respondents suggesting that NgAgo can indeed be optimized for genome...PubMed PMID: 27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo...

...of a single microhomology sequence. For more mechanistic details on HDR and NHEJ, please see the linked... anneal incorrectly, MMEJ may also introduce, substitute, or delete nucleotides in addition to the expected...larger amount of material into the genome. Open questions and alternative systems The ready-made PITCh plasmids...cDNA constructs into this locus. One important question to ask is how the efficiency of PITCh-based genomic...genomic insertion would compare to CRISPR sticky-end insertion using the nuclease Cpf1. Since Cpf1 cuts...-independent knock-ins, but this possibility is still being explored. More broadly, Sakuma et al.’s use...nucleases and their applications. It will be interesting to see what new editing possibilities MMEJ can...

...interested in sharing your science, one of the big questions you'll be faced with is "how?" How are you going...to Rule Them All, consider the following four questions to figure out which method(s) work best for you...You never know what's going to work. After all, a stick-figure internet comic about math probably didn't...Selecting the method Once you’ve answered the above questions, you can create your short list of options and...low data requirements.  No visuals. Variety of hosting platforms can splinter audience. Primarily digital...difficult to convey an entire story or context via still images. Some things cannot be photographed or need...print distribution.  Static. Often requires both artistic and scientific training. Cartoons may be read ...

...developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular interactions...instance, the DBD of the fusion protein shown is still able to bind to the upstream activation sequence... is an example of the former. In this system, histidine-auxotrophic host strains are used to select for...expression of the HIS3 gene, which is required for histidine manufacture and therefore growth on his-deficient...split-luciferase system, intein-mediated splicing reconstitutes functional luciferase only when fusions of the...high number of false negatives in Y2H screens. Testing your reporter system with a pair of proteins that...Y.C., Rajagopala S.V., Stellberger T., Uetz P.  Exhaustive Benchmarking of the yeast two-hybrid system. ...

...AnltWfKuQg pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020 Contest 2: Deck ...temperatures, our first of the season snow flurries, dusting off the winter coats and hats, and our annual Deck...lab space or your work from home space in a fun, festive holiday or winter theme following the rules outlined... pages. Make your WFH space feel a little more festive this holiday season! It is that time of the year... fun, be creative, and enjoy the end of year festivities - however you celebrate! And if you want to see...

...AnltWfKuQg pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020   Deck the Lab: ...Edition Winners In a tie for first place, the Vestibular Lab decorated not just their lab, but their entire... entire floor, and put together a festive video to showcase it all!           View this post on...TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated with dozens of individual virus snowflakes...cookies! 🤗 🤗 🤗 We hope everyone has a safe and festive Holiday season. 🙌 🥂 🎄 🎉 😷 Remember wear a ...

...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid protein...fluorescent signal at baseline voltage), the investigators found that administering these AAVs with a dilute...has developed REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large...their mRNAs, the functional gene product is reconstituted within the target cells (Figure 6).   ...Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing vector... The authors used REVeRT to functionally reconstituting coding sequences for a range of applications...introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity...

...following unanswered questions about CRISPR screen design. Unanswered questions about genome-wide CRISPR...exist to control for these faults. However, it’s still unclear if similar pitfalls exist for CRISPR screens... rates than their non-targeting counterparts, suggesting that safe-targeting guides are more toxic than...closely mirrors that of gene-targeting guides, suggesting that safe-targeting guides better control for...also reproduced several previously observed characteristics that influence gRNA off-target activity: 1)...presents a strategy to control for these effects. By testing thousands of guides for off-target activity, Morgens...

...experiment-based investigation of a hypothesis: proper controls! For every experiment, an investigator needs a ...the effect of the independent variable you are testing. Importantly, they demonstrate the functionality...associated with the independent variable you are testing and not some other unintended factor. Control plasmids...control plasmids would address and answer these questions. Types of control plasmids Part of planning your.... An internal control may be a plasmid that constitutively expresses a reporter protein (e.g., GFP or ...tenet of responsible scientific inquiry and investigation. References: 1. The problem of pseudoreplication...

...genome-editing tool but it could be a fantastic add-on to a diagnostic toolkit, called DETECTR, which has...iceberg as it has been predicted that many others are still unknown. Identifying Cas14 variants In this quest... from the Department of Energy’s Joint Genome Institute for uncharacterized genes that were proximal to...presence of a predicted RuvC nuclease domain, characteristic of CRISPR-Cas enzymes. In contrast to other...middle of the gRNA to be activated. Cas14 in diagnostics By cutting ssDNA but not dsDNA Cas14 is not a...

...are packaged, the physical size of the genomes is still ~4.7kb. But despite this, larger functional transgene...long between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al...site donor and acceptor sequences are used to reconstitute the two pieces of the transgene.The AAV encoding...pre-mRNA of the two halves of the transgene will reconstitute the full-length transgene.  Drawbacks:  Trans-splicing...half of the transgene. The full length gene is reconstituted by either the overlapping or the trans-splicing... template’s integration so that the site is reconstituted after integration; and a sequence of stuffer...since there are two ways for a transgene to be reconstituted, there is currently no one ideal split vector...

...cyanobacterium Synechocystis sp. PCC 6803." Biology open (2016): bio-017129. Hsia, Justin, et al. "Modular...you’re an academic researcher at a nonprofit institution, you can order plasmids covering a wide range...how they’ve been used to date . If you have any questions, please write to us in the comments section.  ...Alexandrov lab at the University of Queensland’s Institute for Molecular Bioscience has provided a solution...library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free expression...The gene set was carefully chosen to perform statistically relevant benchmarking of cell-free expression...

...Zika Virus As always, if you have any questions or suggestions for us, feel free to comment below. Happy...in DNA. The first, contributed by guest blogger Kristian Laursen from Cornell University, is a bit old ...discussed further in this informative post. Interestingly, this technology may make some uses of Site ...Honorable mentions Of course we’ve had many other fantastic posts throughout the month including a first of... Software Engineer Kris Shamloo wrote a very interesting post about how we develop our in house lab management...

...phosphatase treatment, substituting cytidine with 5-methylcytidine, and/or substituting uridine with pseudouridine...behind at the site of excision, this approach is still not considered safe for therapeutic applications...screening cells for the presence of transgenes is still an important control. Additionally, Sendai virus...The typical drawbacks of using transfection will still exist: transfection efficiency is cell-type dependent...by dilution with each cell division, but it can still take several passages for the minicircle to be completely... that ⅔ of subclones generated from EBNA1 iPSCs still had episome DNA present. DNA-Free Methods RNA...reprogramming proteins for 8 hours a week for 6 weeks, and still required further culturing to generate iPSC colonies...

...for students, postdocs, and young Principal Investigators (PIs). We've interviewed Addgene depositors ...Medical School) who took the time to answer our questions.   Advice for success in academia How can we...passion? Even if no one else is excited about it, distinguish yourself with this passion. “Love what you are...laissez-faire; theory or practice; business-like or artistic; tech, anti-tech or pragmatic.” Advice for pursuing..., and George Church for answering our burning questions about how they manage and motivate their labs!...Posts in this Blog Series   Our 3 PIs answer more questions, including: Advice for Choosing a Research Project...