...targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich genomes, but...100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent editing...targeting anywhere in an exon. But things get more complicated if you’d like to use homology directed repair...They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase...test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that the clones...loci displayed a broad range of PAM sequences, indicating they may be less predictable than SpCas9 off-... that we have only scratched the surface of modifications that can be made to Cas9. It is a pleasant surprise...

... the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's important...2000. The Cell: A Molecular Approach. 2nd Edition. 3.  CureFFI.org. Cell Biology 04: The Secretory Pathway...functionality can be gained by understanding the location and transport of the protein within a living cell...to observe membrane trafficking events in vivo. Locate your protein and see who it is hanging out with...potential overlap, it is possible to determine the location of proteins within complex structures. In order...way it is important to choose the appropriate quantification methods and tools. It is usually not enough...intracellular pathogens In addition to tracking the location of proteins it is also possible to follow the ...

...success of CRISPR-Cas9 across multiple model organisms (3) lies in the versatility of its delivery methods. ... "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo: Genome-editing by a DNA-guided enzyme." DNA-scissors...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...survey. When asked if they could detect indels (an indication of gene disruption ability) with NgAgo, one respondent...could not achieve indels with the NgAgo system indicates that it is very tricky to use and fussy to optimize... S. Lander, and Feng Zhang. "Development and applications of CRISPR-Cas9 for genome engineering." Cell...Addgene.org Request NgAgo Find CRISPR Resources Catch Up on Your CRISPR Background ...

...be controlled by blue light (Bubeck et al., 2018). 3. Reduce cytotoxicity of CRISPR-Cas editing in tissues...Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv 3:e1701620 . https://doi.org/10.1126/sciadv.1701620 ..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...viral particles that have the gene deletion could replicate when challenged with the S. islandicus CRISPR-...B, Bondy-Denomy J (2020) Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies....light-inducible Cas9 activity? Check out these these plasmids Catch Up on Your CRISPR Background with Our Guide Pages...

...carrying the desired genetic modification integrates into the genome (Figure 3). Here, the chromoprotein ...  Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP and tsPurple genes... sequencing are more definitive, but visual identification is a good place to start before taking on more...resources are limited or for bioart and teaching applications. Find a chromoprotein for your experiment! ...of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes. Applied and Environmental...

...Ras pathway. The term “Ras” collectively refers to 3 proteins (H-Ras, K-Ras, and N-Ras) encoded by a family...cell 25.3 (2014): 272-281. PubMed PMID: 24651010. 3. Ledford, Heidi. "The ras renaissance." Nature 520.7547... millions of people die from cancer-related complications each year. Despite these grim statistics, new...any of these genes through several different applications such as fluorescent protein fusion studies and...Lemieux is a Scientist at Addgene with interests in education and scientific writing.     References 1. Martz...Martz, Colin A., et al. "Systematic identification of signaling pathways with potential to confer anticancer...

...binds EQKLISEEDL (pronounced “equi-kli-seed-le”). (3) Tag all the things!   Myc1-9E10 and the Myc-...mapping functional domains of Drosophila hsp 70. Embo J 3:3087–3093. https://doi.org/10.1002/j.1460-2075.1984...used in way more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal... by its antibody without additional chemical modification steps and when the epitope was within the tagged...

...industry and how delegating can empower people Chapter 3: How Crystal advanced at Novartis, and how to prepare...Intro Chapter 1: Who is Crystal Shih? Crystal's educational background and position at Novartis. Chapter ...Chapter 7: How Crystal developed her science communication skills and how they've helped her at Novartis...

...ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half an hour can help keep...Journal of applied phycology27.6 (2015): 2321-2332. 3. Contamination, Ileana S., John P. S., Basic Methods...results. Most cell culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red))...Anything between orange and light orange usually indicates that the cells have grown enough, are making their...

...adenovirus genes which help mediate AAV viral replication; and 3) the transfer plasmid which encodes a transgene...ITR-containing plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours...which provides the AAV replication (rep) and capsid (cap) genes. AAV replication uses the host’s polymerase...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ... that aids in the homologous recombination of duplicated regions like the ITRs. Restriction digest screening...consider performing PEG precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV...

...Professional Certification Institute (GPCI). GPCI evaluates a candidate's expertise (a minimum of 3 years experience...the organization(s) you serve time and resources. 3. Get a mentor, find your people Find someone who is...development, certification, degrees, foundations, coaching and even Twitter Chats dedicated to the Grant...the candidate application process, the candidate’s ability to write a grant application through a written... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...

... enhancers.  3. Measuring temporal changes in chromatin interactions A third application for CAPTURE 2.0...expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses delivering... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...could only detect chromatin interactions at one location in the genome at a time (Liu et al., 2017). Recently...at once (Liu et al., 2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original...biotin ligase a gRNA(s) targeting a single genomic location of interest  When these three components are ...

...prioritize investments over the next 3, 5, or 10 years. Complicating these challenges is the fact that they... Consulting firms deploy case teams, usually with 3-6 consultants, who work on analyzing the situation...advancements. Consultants can help these teams develop communication strategies, or understand, and publish on the...success. Or the client might need help with the intricate task of designing a strategic vision for a portfolio...chemotherapies, or developing innovative mobile phone applications and services that help diabetic patients manage...knowledge from previous consulting experiences, educational training, and conferences, and synthesizing this...that gives the client a defined path forward. Communication is key as ideas must be relayed quickly both...

...had therefore been trafficked between locations). Figure 3: A) LOV-Turbo becomes active when illuminated... h, and PMA activation induces cFos::GFP-iPAK4 at 3 or 6 h. Scale bar 5 µm. Images adapted from Lin et...Figure 1: Choosing the best PE6 variant for your application. Image reused form Doman and Pandey et al., 2023... CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features of DIRECTED...approach for experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D...delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467...interactions or map a local proteome, but some applications have been challenging or inaccessible. The Ting...

...enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals which...helps us to locate interesting addresses in the genome. Let’s now examine how we can locate powerful stretches..., start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the ...for the genome" techniques Technique What It Locates ChIP-seq Proteins (for our example later, histones...therapeutics. We’ll have more to say about clinical applications of CRISPR in the next section. In order to denote...’s associated histone proteins K4 and K27 – locations of the amino acid lysine in H3 me1, me3, and...aceylation, respectively (these are chemical modifications, or functional groups, found on lysine) Putting...

...propagation along dendrites for the first time (Figure 3). The team also used AAV9 to deliver ASAP5 to the ...microscopy at depths up to 600 µm. Figure 3: ASAP5 for voltage imaging. A) ASAP5 selected for ...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration... an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment.  Figure...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted with permission...

...31668202421', style='text-align: right;' %} Figure 3. An animated map showing the plasmid orders and new...were zero plasmid requests. Afterwards, from 2/24/20–3/15/20 (the supposed end of lockdown), request activity...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...14%). Spikes in plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests...effect on plasmid requests Before we began any sophisticated analyses, we first visualized our data in various...orders and new COVID-19 cases for each week by location. We observed that as expected, plasmid orders ...independence assumption of the Poisson process. This modification also eases the interpretation of the parameters...

...PMID 25282355. PubMed Central: PMCID: PMC4317352. 3. Beisel, Chase L., Ahmed A. Gomaa, and Rodolphe Barrangou...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such... and medical organizations are working to help educate the public about proper antibiotic usage. Although...mammalian viral vectors in that the vectors are replication-deficient. Thus, a very large number of phagemids...CRISPR antimicrobials combines them with both replication-competent phages and antibiotics. Phage therapy...

...sequence only functions at the extreme C-terminus [3]. You must therefore determine your cloning strategy...PMID: 19819147. Pubmed Central PMCID: PMC2784028. 3. Munro, Sean, and H. R. Pelham. "Use of peptide tagging...compartments undergo significant post-translational modifications including glycosylation, disulfide bond formation...are equally susceptible to these non-native modifications, which can all potentially affect function [...N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering the protein... non-optimized parental proteins. Our recent publication illustrated the quantitative increase in fluorescent...and Jack Griffith, her research focuses on the replication machinery of Kaposi’s sarcoma-associated herpesvirus...

...and makes your experimental setup less cumbersome. 3. Portability across multiple systems Terskikh et al...PMID: 19889833. PubMed Central PMCID: PMC2801723. 3. Verkhusha, Vladislav V., et al. "An enhanced mutant... (green or blue) within a population of timer indicates the age of the pool: the higher the ratio, the...-FT-N1 pTRE-Slow-FT pBAD/HisB-Slow-FT -  Applications of fluorescent protein timers FP timers have...biological event (trafficking to a subcellular location, start of gene expression, development of a cell... insulin-secreting beta-like cells." Nature communications 7 (2016). PubMed PMID: 27063289. PubMed Central...Learn More About Fluorescent Protein Aggregation Catch Up On Your GFP Basics Get Advice on Choosing Your...