...By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create...mutation inactivates the RuvC domain, so this nickase cleaves only the target strand. Conversely, the H840A mutation... in the HNH domain creates a non-target strand-cleaving nickase. Instead of cutting both strands bluntly...distances (40 - 130 nt). From these results, it’s clear that editing is much higher in the PAM-out configuration... At the AAVS1 locus, the two nearest gRNAs had cleavage sites 12 nt and 13 nt away from the target site...wide margin, exhibiting >20% repair efficiency. Clearly, a nickase strategy can expand the targetable region...post. Additional Resources on the Addgene Blog Learn about Homology Directed Repair 3 Tips to Improve...

...Careers in Science Communication blog series, you’ll learn about Caitlin Runne-Janczy, a Product Development...While her love of educational science writing is clear, Caitlin’s career plans weren’t concrete when she...what I wanted to do, but knowing that I wanted to leave the bench.” Her path to figuring out her career ...writer.” At the end of her postdoc, Caitlin had a clearer idea of her career goals: “My plan was to go into...for part two of Caitlin’s interview where you can learn what it’s like to work for a science education company...series!  Additional Resources on the Addgene Blog Learn more about alternative science careers here Be like...while in grad school Resources on Addgene.org Learn more about careers at Addgene ...

...non-academic career Learn more about alternative science careers here Resources on Addgene.org Learn more about...series was written for selfish reasons: I wanted to learn about careers in science communication. When I started...away-from-the-bench career option. So if you’re interested in learning more about science communication careers, you’...was a science writer, reached out to me. She was leaving a writing position and looking for someone who ...s paid off. Work is never boring. “I just keep learning on the job. It keeps it interesting.” And while...topic, but I haven’t met any.” A few other tips: “Learn HTML, write a lot to make sure you don’t hate it...

...depurination, the hydrolysis of the glycosidic bond leads to the release of a purine (adenine or guanine) from the...DNA are repaired by base excision repair, but can lead to mutations in single stranded DNA undergoing replication... from E. coli is the presence of the protein endonuclease I encoded by the gene endA. Nowadays, the most...gene has been removed/mutated to neutralize the nuclease activity. For strains that still carry this gene...introduced in the purification protocol to remove endonucleases activity. For these reasons choosing the correct...most downstream processing. On the other hand, nuclease free water is a great option because it provides...

...professional Addgene is a thought leader in scientific sharing, leading by example to increase sharing in...Resources on Addgene.org Learn More About Our Mission Meet the Addgene Leadership Team Get the Latest News...arts organization. Many people are surprised to learn that Addgene is officially filed, recognized and...is a nonprofit? The term "nonprofit" is a bit misleading. Nonprofits can generate revenue (as Addgene ... Xuan Additional Resources on the Addgene Blog Learn about Our Collaboration with Fellow Nonprofit Seeding...

...its endonuclease activity to generates a ssDNA substrate which is then nicked by its endonuclease domain...engineered Learn about the parts of the AAV transfer plasmid  Resources on Addgene.org Learn about AAV...of Rep78 and Rep68 contains a DNA-binding and endonuclease domains, all four Rep proteins share a common...virion assembly, the role of MAAP has not been clearly defined yet. Now you’ve got a basic understanding...AAV RepCap plasmid! Check out this blog post to learn more about the AAV transfer plasmid! References...

...Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts in your experiments. Read this ...Professor at Albert Einstein College of Medicine, to learn how problematic oligomerization can be and how to...that occur during development. Read this post to learn more about recombinase-based state machines and ... Green (both formerly at the Wyss Institute) to learn how these amazing tools may be used to rapidly diagnose...you can use to study basic virus biology while leaving virulent components behind. Minigenomes are stripped...and transcription as well as reporter genes. To learn how you can use minigenomes in your research, check...

...s antibodies are plasmid-based and recombinant. Learn more about recombinant antibodies here. Finally,...Finally, Addgene is a nonprofit that connects colleagues and is invested in improving research infrastructure... to maximize the reach and impact of your work. Please email [email protected] if you would like to ...plasmids via Addgene! Email [email protected] to learn more or get started.  If you have suggestions about...ideas for new materials to add to the collection, please let us know by filling out this suggestion form... will find useful antibodies for your research. Please consider sharing your experiences with antibodies...Resources We would also like to acknowledge our colleagues at the Developmental Studies Hybridoma Bank, ...

...the Addgene Blog Learn more about FRET Tips for Using FRET in Your Experiments Learn more about Fluorescent...brightest since it had an emission near 600 nm – a clear demonstration of why a BRET reporter can improve...Many intracellular sensors of events like calcium release have been made using fluorescent resonance energy... molecules. FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting...wavelengths, excitation of the optogenetic regulator leads to spurious activation of the FRET sensor. Researchers...et al. (7) wished to trigger intracellular Ca++ release with an optogenetic receptor, melanopsin, requiring...#83926). Live-Cell Voltage Sensor  Ingaki and colleagues (8) were interested in using photoactivatable...

...nature of non-homologous end joining (NHEJ) often leads to the generation of indels and thus frameshifts...activation) and CRISPRi (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter...landscape of mismatches that can nevertheless still lead to activity has been established, and can be used...that the consequences of off-target activity, at least for the experimental conditions used, led to no ...the generation of truly isogenic cell lines than nuclease-mediated off-target effects.” Further, large-scale...al., 2019). Again, the experimental strategy is clear: for any gene of interest, one should require that... Liu DR (2020) Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors....

... about roles in this field, most jobs needed at least 3 months experience working in a developing tropical...positive experience. I enjoyed helping people and learning to explain concepts in ways that people from another...traditional medicine couldn’t fight effectively. I had to learn how to cope with a different climate and culture...work became monotonous very quickly; as soon as I learned how to do the different tests and became efficient...where one drug does not work for all patients, leading to very high mortality. I really loved the fact...cannot be as adaptive. I was also surprised to learn that I enjoy the customer service side of my job...vital because otherwise I’d lose touch with my colleagues and have misunderstandings.  Working long distance...

...bound lipoproteins Article contributed by Leah Schwiesow A leading cause of death worldwide, cardiovascular...ever like to write about a recent plasmid deposit please sign up here.        A retroviral mouse CRISPR...genetically. To solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library ...lipoproteins through the circulatory systems, but can also lead to the formation of atherosclerotic plaques. ApoB...through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman et al...-LP size and concentration, Steven Farber and colleagues at the Carnegie Institute for Science have developed...

...of a paper is the one that you need to spend the least time on—just put down a basic outline and refer ...figure them out. But, when they come to light, it’s clear that what the researchers thought was a problem ...role, and we take our responsibility to push for clear communication of science very seriously. That’s ...and transparency of methods sections, including clear information about reagents, and why other publishers...want to be? The part that is still missing is a clear alignment of incentives. Right now, researchers ...evidence of sharing for grant renewal—but there is clearly room to do more. If we could really instill a culture...Her current responsibilities encompass overall leadership of the Cell Press editorial department, a team...

...often a hard question to answer if you plan on leaving academia, but don’t quite know what you do want...goal, and when you realise that your goal might not lead to a career you actually want, you can feel lost...Journal Editor Science Marketer Software Developer Learn Science Communication Skill from a Student Group...in the media and in films, which can often be misleading. Key aspects to determine are working hours (... the job require public speaking? Will you be leading a group or working as part of team? Will you be...application. Additional Resources on the Addgene Blog Learn How to Experiment with New Careers while in Grad...Koeris' Journey from Grad Student to Entrepreneur Learn to Network and Make the Contacts for Your Dream ...

...describe a process that begins with cell lysis to release AAV particles with subsequent purification steps...also possible for AAV vectors to be spontaneously released from the producer cells into the supernatant during... are exosome AAVs? In 2012, Casey Maguire and colleagues from Massachusetts General Hospital and Harvard...took a closer look at the AAV vectors that are released from cells during production (Maguire et al., ...the exo-AAV isolation protocol by Maguire and colleagues, other researchers have tried to modify it to...possibly improve transduction properties. György and colleagues found that the association of AAV particles with...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...miscommunication, a lack of leadership, and inappropriate tools or writing approaches can lead to frustration, delay...Weill Cornell Medicine. Discussing data with colleagues can be more difficult during the pandemic era...can contribute and sketch on the board without leaving Zoom. The annotate function can also make the screen-sharing...cultural backgrounds.” This work outlines the need for clear communication, fair credit through authorship, and...ways of working, reconnect as human beings and colleagues, eventually encouraging purposeful and creative...Medicine in NYC and author of the book “The Salmon Leap for PhDs: Swimming upstream a career from academia...

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... we've heard over and over from our users: "Please, please improve your plasmid maps!" After thoughtful... here to see the new map in action). Read on to learn more about the improvements we’ve implemented and...extraneous ORFs that cluttered our old plasmid maps. Clearly Annotated Features, Enzymes, and Primers - The ...easier for you to share plasmid information with colleagues, or explain plasmid features to a lab member ...Once you’ve identified a plasmid that you want to learn more about, updates to our sequence analyzer will...hover over any feature, enzyme, primer, or ORF to learn more information about it. For example, if you hover...

... Sample6 has changed focus and names, swapped leadership, and grown to 25 members.  What is Sample6?...competing in business plan competitions. After much learning, the focus of the application was changed, but...best, wins. He explained that entrepreneurs must clearly communicate their idea at various levels of complexity...industry vary drastically, he reassures that one can learn it by reading a broad variety of materials and constantly...million. The biggest challenge during this time was learning to communicate effectively to non-scientific audiences...are essential to the biotech startup puzzle: Nucleation of the team. The team is key, and the initial...raise money and/or validate an idea or product? Please let us know in the comments section! And read on...

...often terminate with a poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.    ...site for integration is TA dinucleotides and it leaves behind the CAG DNA footprint from its terminal ...terminal sequences at the excision site after cleavage by the transposase. It has a cargo capacity >100 kB, although... TTAA and unlike other transposons, it does not leave behind a DNA footprint sequence after excision. ...   Applications of transposons Now that you’ve learned about some of the popular transposon systems, let...DNA into the pronuclei of fertilized eggs, which leads to random incorporation of that sequence into the...ijms22105084 Additional resources on the Addgene blog Learn about the INTEGRATE system for bacterial genome ...