...disciplines, the base technology itself is constantly improving. Cas9 variants have been modified for genome editing...), revealed that these mutants do not cause off-target effects at unanticipated sites and, as predicted...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target... that CRISPR/Cas9 genome editing can result in unwanted changes at non-target sites. Means to decrease...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA ...substitutions throughout the groove in 32 separate Cas9 mutants. When tested for their ability to cut the EMX1 ...embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target activity of wt Cas9 while...

... whether respondents saw them as not important to very important. In addition, we also explored the barriers...save them significant amounts of time and effort and give the lab a serious competitive advantage.  Many...functionalities that are hard to imagine on paper, such as instant search through all saved data and automated writing...survey respondents indicated that cost was a significant barrier to ELN adoption. This includes financial... study, respondents were also asked about how important ease of use is for them (if they are to switch...ELN choice, with almost 80% rating it as very important. One comment reflected the desire for a flexible... category;”. The study also showed that It is important that an ELN supports all types of data, which ...

... This emphasizes the advantages of bona fide pSer-incorporation into recombinant proteins as an alternative...is a particularly important control when creating a phosphoserine-incorporation mutant for a residue that...low abundance, and thus difficult to purify in quantities necessary for study. Moreover, the ability to...usually depends on tools such as phosphospecific antibodies, which aren’t available for the majority of documented...it’s possible to generate non-phosphorylated recombinant protein and perform in vitro kinase reactions...establishment of a system in which purified, recombinant protein could be produced with site-specific ...lacked a classic cysteinyl tRNA synthetase, despite abundant incorporation of cysteine in their proteomes (...

...diseases caused by Arg-mutant proteins, but there is currently no evidence of Arg-mutant protein rescue in ...rescue multiple Arg-mutant proteins in living baker’s yeast cells, including Arg-mutant yeast homologs of... the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases through...disorders of glycosylation Paula Videira is an Assistant Professor at the Faculty of Sciences of NOVA University...other. Indeed, nearly 20% of the single missense mutant alleles that cause human disease change Arg to ...rescues the function of even a small fraction of Arg-mutant proteins could improve health outcomes for millions...that the in vitro functions of 11 different Arg-mutant enzymes could be rescued by supplementation with...

...scientists have co-opted antibodies for tagging purposes. Different antibodies bind different protein sequences...tag their proteins of interest. Antibody based probes, such as antigen-binding fragments (Fabs) (Porter...together. By combining two optimized elements of an antibody probe, scientists can now more easily visualize...amplify and image for long periods of time.  Are antibody based probes a solution? To address these limitations...HA frankenbody To combat the issues with these antibody based probes the Stasevich lab used an alternative...alternative strategy to develop a genetically encoded antibody based probe that detects the widely used HA epitope...complementarity determining regions (CDRs, or loops) of an antibody that recognize and bind specific epitopes (Ewert...

...protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR proteins are a highly diverse...our blog post: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems.  Listen to the anti-CRISPR podcast ...Light-Dependent Anti-CRISPR Bubeck et al., 2018 CASANOVA LOV2 from A. Sativa AcrIIA4 anti-CRISPR protein...Cas9 activity. The use of nickases, prime editing, anti-CRISPR proteins, and other techniques all aim to...gene expression. Both of these techniques take advantage of light-induced dimerization to activate their...C714, generating paCas9-2. This change did not significantly alter the system’s efficiency at generating ...photoactivatable repressor (dCas9). The activity of all variants was reversible and repeatable. Since these initial...

...found that exo-AAVs were more resistant to neutralizing anti-AAV antibodies. This is particularly useful... to reach, and are more resistant to inactivation through anti-AAV antibodies. References György, Bence...therapeutic applications in vivo where endogenous anti-AAV antibodies often neutralize the therapeutic efficacy...spontaneously released from the producer cells into the supernatant during the production phase. However, this has...researchers discovered that these particles have some advantages over the conventionally purified AAV vectors....centrifugation steps to separate cell culture supernatant containing exo-AVVs from producer cells, and ...particles with membrane vesicles protects them from antibody recognition and inactivation (György et al., 2014...

...AAV plasmids contain several important elements, but among the most important are the inverted terminal ... ITR integrity and prevent unwanted mutations?  During propagation, mutants rapidly replicate in a growth...Next-generation sequencing is also helpful in quantifying ITR mutants since analysis of individual reads allows...Adeno-associated viruses (AAV) are an important biological tool that allow scientists to express a gene...and the ITRs are retained with the gene in a recombinant AAV (rAAV) vector (Figure 1). Everything else...outside the ITRs gets left behind.    Figure 1: Recombinant AAV vector with ITRs that flank the gene cassette...modifications and types  Since ITRs are one of the most important elements of AAV and rAAV vectors, modifications...

...might want to pick a yeast strain best suited to it. Putting K. phaffii to work   The yeast giants - S....alternative model yeast strain and discuss why you may want to consider a yeast off the beaten path.   Yeast...have a human counterpart, enabling study of human-relevant pathways (Liu et al., 2017). Beyond human relevance...well as glucose or glycerol). K. phaffii is only distantly related to S. pombe and S. cerevisiae; evolutionarily...attention for its powerful promoter of a gene important for methanol usage. This gene promoter, AOX1, ..., was co-opted for the expression of recombinant proteins by the pharmaceutical and biotech industries...efficient at protein secretion, making harvesting recombinant proteins a cinch. This is because it doesn’t ...

... AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining access ...sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor for PHP.B. Read ...PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J mice is likely...screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared...efficiency, but at a log lower dose than PHP.B.  A Ly6a variant is linked to PHP.B CNS permissiveness Both the ... labs determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach...

...incompatibility with antibiotic-resistance plasmids, scientists have expelled antibiotic resistant plasmids from...encode a negative regulation system involving antisense RNAs or iterons that inhibits replication when...number in the cell falls too low (Novcik, 1987).  Antisense RNAs encoded on the plasmid either indirectly ...plasmid increases, increased amounts of these antisense RNAs inhibit plasmid replication.   Figure...incompatibility going to affect my cloning project?  If you want to clone two plasmids within the same bacterial ... uncommon, or perhaps a synthetic biologist who wants to develop a plasmid-based tool that is easy to ...Using plasmid incompatibility for therapeutic advantage Although it may seem like plasmid incompatibility...

...nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...harness those features to create broad host range recombinant vectors.  A plasmid’s host range is determined...phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids...host-independent   Building broad host range recombinant vectors Replicons, which consist of a plasmid...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...methanolicus, Methylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas...Rhodospirillum spp., Shewanella spp., Thiobacillus spp., Xanthomonas campestris IncQ RSF1010, R300B, R1162 ...

...by guest bloggers Erik L. Snapp and Lindsey M. Costantini. "You underestimate the power of the Dark Side...non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general... the cytoplasm? Or more troubling, what if a significant fraction of the fusion protein molecules fail...population is not readily apparent and can confound quantitative imaging experiments or even negatively impact...fusion based on your experimental needs. Equally important, before cloning you must consider whether and ...resident proteins within these compartments undergo significant post-translational modifications including glycosylation...misfolded dark FP fusions including erroneous quantitation of fusion protein levels. It remains unclear...

...The FLAG antibody has been well characterized and the system allows you to add this same antibody to any...known to have good antibodies. The results look almost identical. Addgene: What advantages does your system...established method called ChIP-seq. ChIP-seq relies on antibodies to recognize and bind transcription factors directly...the transcription factor. It also gives cells antibiotic resistance to neomycin so that you can select...of the proteins inside a cell have a commercial antibody against them. There’s also a question about how... solved in this CRISPR system with a universal antibody. It’s an epitope not found in human or other cells...transcription factors, but there are many reasons you might want a system like this. It’s actually surprising to ...

...Salary –not relevant to most postdocs. Commercial successes in the performing arts- not relevant at all. ...options discussed in this post benefit from the applicant thinking about the process early, keeping good... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a... cap subject H-1B (last year less than 30% of applicants received sponsorship) then he or she can start... facility management, collaboration management, grant administration, clinical operations, and so on. ...1B petitioners are exempt from the cap if the applicant works at institutions of higher education, a related...US Immigration officers use to determine if an applicant qualifies for the National Interest Waiver. The...

... our ready-to-use recombinant antibodies service, part of the Neuroscience AntiBody Open Resource (NABOR...library of antibody educational resources, including our new Antibodies 101 eBook, the Antibody Guide, and...could use the same principles to provide recombinant antibodies, another plasmid-based product that, with...front of us. So in our last celebratory moment, we wanted to walk through some of our favorite milestones...designing a website users could easily navigate. This meant we could grow accessibility alongside our repository...so easy!” — Nina Jain, Harvard Medical School. We want to give a special thanks to the early-adopter scientists...beginners” on r/labrats, Reddit.  Part of that work meant facilitating the Material Transfer Agreement process...

...are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made by introducing...contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology...jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...that generates the chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and ...Cranfill et al., 2016 and FPbase.org). A number of variants remain fluorescent under acidic conditions, with...N-terminus, but this hardly reduces its size. The substantial size of GFP may interfere with the activity or...

... community which is really important to her. Addgenie Shreya Vedantam with our resident dinosaur...school. QC Scientist Alyssa Cecchetelli knew she wanted to get her PhD. “Going back to school was a pretty...in Addgene since he started, said, “the thing I wanted more of [after undergrad] was to learn and speak...may open doors for you, but depending on where you want to go in your career you may not need those doors...her first job at Addgene as an Order Processing Assistant. After a few months, she left school when she ...Technician. “I was so tired of being in school so I just wanted to work,” she said. “School will always be there...mean you’ve failed. “I got a job as a research assistant in a lab at the university that I went to. Making...

...Notably, the SpRY variant didn’t display higher off-target effects than SpCas9, a pleasant surprise since... PAM requirement, and how these proteins are advantageous in today’s genome editing landscape. The need...anywhere within the RNA will do. Similarly, if you want to knock out a gene, a frameshift mutation introduced...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative...looking for more pan-genome PAM flexibility, you’ll want to consider one of several Cas9s that recognize ...compatibility. This alternative PAM requirement adds a significant amount of flexibility compared to a strict single-compatibility...rounds of engineering, they developed the SpRY Cas9 variant (Walton, et. al, 2020). SpRY uses NRN PAMs and ...

...recombination software looks for and quantifies the presence of recombinants in recombinase-dependent sequences...experiments to confirm their findings. But no scientist wants to repeat an experiment because of poor reagent ...production process? These questions are especially important when producing multiple vectors in parallel because...serotype of the packaged DNA and look for potential contaminants. The analysis is a 2-step process using Geneious...total reads are most likely to correspond to the contaminant. Each contig is then BLASTed against the NCBI...contaminated. For AAV-68544, the best match for the contaminant is Addgene plasmid #44362. Method validation,... were easily able to identify AAV-44632, the contaminant we mixed into the original AAV sample at 5%. ...