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...retinal ganglion. Rather than using naturally occurring promoters to drive transgene expression, the Roska...one type of cell, with the remaining expression occurring in only Müller glia cells, a common contamination...retina.  Find the AAV synthetic promoter plasmids! Comparing synthetic promoters performance in mouse, monkey...targeted cells in monkey and human retinas. By comparing the performance of promoters across these three...be paired for targeted expression. This same pairing of synthetic promoters were also used to target...

...their promises, Wet-blanket negativist, who can bring down the morale of a group with their pessimism,...communicate their ideas confidently while also considering the needs of other people. Some students and ...Regardless of your supervisor’s management style, bring challenges to his or her attention as soon as you...signed, or manuscripts that need to be reviewed, bring them to the meeting. This will make it easy for ...problem by stating the facts and explain how it is interfering with your work. Avoid talking about your emotions...

...special considerations. Conventional flow cytometry During conventional flow cytometry, light emitted by a...distinguish these two fluorophores from one another during spectral unmixing.     Figure 2: The ...a three color panel or a 40 color panel! When preparing a spectral flow experiment and panel, there are...telling exactly how many Cy7 molecules are added during that particular batch. This can present particular...upon antibody binding, leading to discrepancies during unmixing. Additionally, it is critical to include...

...something done not by people wearing a lab coat, but instead by people wearing business suits discussing ...transferable skills while working in the lab and by offering advice on areas where you might need some help...you are negotiating. Then take a moment before, during, or after a negotiation to think about the situation...experience in the area I want to explore while also ensuring that my work gets completed? I want my company...

...perform functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided... endonuclease NgAgo! We'll discuss how NgAgo is faring with users in a minute, but, to start, let's review...cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering methods have their own pros and cons (2). The...protein to load an ssDNA guide. It is possible that, during translation, only a small fraction of nascent NgAgo...Development and applications of CRISPR-Cas9 for genome engineering." Cell 157.6 (2014): 1262-1278. PubMed PMID: ...

...Introduction There are three primary methods for repairing DNA after a double-stranded break. HDR copies ...mutations seen with TALENs and CRISPR. MMEJ is active during M and early S phases, when HDR is inactive, and...Non-homologous End Joining Read About Other Genome Engineering Tools like Sleeping Beauty Mutase Find the Ideal... genome engineering... Mary Gearing...

...heterozygote into a homozygote and ensuring that all future offspring inherit the endonuclease gene. As...As we pointed out last year2, delivering a DNA cassette encoding the cas9 gene and single guide RNAs (...shared by everyone, he is deeply concerned with ensuring that gene drives and other collective technologies...selfish genes as tools for the control and genetic engineering of natural populations. Proc. Biol. Sci. 270,...

...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...producer cell membrane, and acquired as an envelope during exocytotic budding, as the viral particle emerges...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription... integrated by the viral integrase protein (IN) during step 3 (King et al., 2008) (Hu & Temin, 1990). ...

... the Addgene blog Learn more about plasmid sharing during COVID-19 Resources on Addgene.org Find plasmids...False, type='scriptV4', hide_playlist=True, viral_sharing=False, embed_button=False, autoplay=False, hidden_controls...to follow an exponential decay model, with dips during weekends and significant holidays. Figure...likelihood by the corresponding factor 𝜙z if t is during event z. The "events" we considered for our model...

...presence of antigens (such as viral vectors) without triggering a destructive inflammatory immune response. At...after the cloning of the RPE65 gene, a naturally occurring animal model, the RPE65−/− dog, was characterized...retinal neurons. Nowadays a toolbox of naturally occurring and engineered AAV serotypes that further expands...being done to further improve the technology: engineering of new capsid variants, designing expression ... optimize transgene levels, and large scale manufacturing of viral vectors to support trials for prevalent...

...If you're interested in sharing your science, one of the big questions you'll be faced with is "how?"...for longer educational videos, and Twitter, for sharing updates, new deposits, and new blog posts!  ...  of proficiency.  What kind of content are you sharing?  Sometimes your subject matter will drive the ...fair, or a semester-long after school program? Comparing your desired outcome to the limitations and potential...response. High connection levels and interaction. Can bring in things to be physically interacted with. Public...

...suppose you have a piece string that’s 10 meters long. You twist and roll the string into a tight ball. Now...in a way that brings linearly distant regions close together. gRNA Selection and Filtering for Off-target...Now the two ends of the string, instead of being 10 meters apart, are mere millimeters apart. Similarly...

...bacteria and prevents sensitive bacteria from acquiring resistance genes. Yosef et al. suggest that it...not previously infected by the lysogenic phage, rendering the population sensitive to antibiotics. Created... Lemire, Diana P. Pires, and Timothy K. Lu. “Engineering Modular Viral Scaffolds for Targeted Bacterial...Addgene Blog CRISPR Methods for Bacterial Genome Engineering Treating Muscular Dystrophy with CRISPR Gene ... Mary Gearing...

...resulted from excision by uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the...off-target mutations than those derived from naturally occurring cytidine deaminases. Recent efforts have also ...compared to ABEmax. In an alternative approach, engineering and directed evolution of the TadA domain has...) This post was originally written by Mary Gearing in August 2016 and was updated by Jennifer Tsang...Y., Guan, Y., Siwko, S., … Li, D. (2023). Re-engineering the adenine deaminase TadA-8e for efficient and...applicability of base editing through protein engineering and protein delivery. Nature Communications, ...

...studying chemical engineering. Mengtong "Tom" Duan is a PhD student studying bioengineering. They are both...smaller genetic footprint, and nonlinear acoustic scattering. The best candidate for this was the Anabaena...was sufficient to saturate expression. When engineering a cell line, cotransfect the mARG cassettes 2...acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria. Nat Biotechnol...

...pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species... cell stress, and RNA size distribution. When comparing LwaCas13a to shRNA, they found that LwaCas13a ...vivo RNA imaging. In short, the Cas13a molecule brings a much anticipated element of diversity to the ... the CRISPR field. We feel that the culture of sharing within the CRISPR community is an essential part...scientific community. Note: Cary Valley and Mary Gearing contributed to the update of this post. References...

...editing reporters that can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time...results in two DSBs that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using...editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells...real-time, it relies on DSBs which is not ideal for monitoring single base edits (St. Martin et al., 2018). ...the Harris Lab circumvents the need for DSBs in monitoring on-target DNA editing efficiency of APOBEC-Cas9...

...viral genome). Empty viral capsids can be formed during viral production depending on the  experimental...usually report? Addgene and other viral vector manufacturing facilities report the physical titer of a viral...to two-fold. How can we get more reliable AAV titering? For our viral service, we currently titrate AAV...different sources in the same experiment, consider retitering both AAV in your lab. The absolute titer values...Addgene, we don’t reserve lots, but if you’re reordering the same virus feel free to email us and we can...

...expand and exploit CRISPR, both of which we're featuring in today's blog. Cas7-11 Development and Discovery...alongside some creative (and significant!) protein engineering. They combined Prime editing with an integrase...potential applications. But they weren’t done engineering quite yet – surely, they thought, the integration...optimized the specific attachment sites via sequence engineering, enabling even more efficient integration activity...: Adding Precision and Flexibility to CRISPR Engineering CRISPR 101: RNA Editing With Cas13  CRISPR Cheat...

... data. Although it may be useful to selectively bring particular items into an ELN – e.g. an image that...the integration with protocols.io, the protocol sharing repository. As illustrated in this video the integration...RSpace becomes part of a workflow that enhances the sharing and reproducibility ecosystem protocols.io makes...help with searchability, record-keeping, and data sharing, not all of them support reproducibility in the...Reproducibility Read all blog posts about scientific sharing Resources on Addgene.org Find protocols of molecular...