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...expression (Sapozhnikov and Szyf 2021).    Figure 3: A simpler approach - dCas9 blocks DNMT1 methylation...work well, and up to 10 base pairs away (especially 3’ of PAM) might work too.  High dCas9 and gRNA expression...approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   Figure 1:... when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9....for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34, 1060–1065. ...

...passion and pay attention to the details. Chapter 3 - Answering interesting biological questions using...Twitter and has helped us update many of our educational resources. Listen to hear all about Professor...speeds science. Chapter 8 - The importance of communicating research findings through social media and other...

...biological study without her or her family's knowledge. 3. Fossil Rock Anthem One of Tom's earliest and most...advice on how to dive into new forms of science communication. Click here to listen to the interview with ... from Addgene.      Check out more science education tools like Verse Two and Fill in the Planck at...with us. Tom McFadden is a YouTuber and science educator looking for creative ways to implement the next...next generation science standards and communicate science. Learn more about Tom on his youtube channel ...Community Check out All of Our Podcasts Find Great Educational Content in our Plasmids 101 Series Additional...

... to antibody response 2) very high immunogenicity 3) and long-lasting immune response even after a single...their use in other clinical applications. In the last decade, viral applications in cancer treatment and ...s unpack the myriad number of emerging viral applications and give you some recommendations for putting...putting viruses to work. Viral applications Viruses work by infecting a host cell (the target of viral infection...expressed. It’s important to note that for all the applications we discuss, there is more than one type of virus...retrovirus, AAV, etc.). When you’ve decided on an application, our Viruses as Biological Tools blog can help...viruses and pick one for your experiment. Research applications Gene expression control Researchers are always...

...degradation of endogenous proteins (notably, BRD2/3/4, CDK9, TRIM24, FLT3, BTK, and ALK) by linking small...with dTAG-13 at doses as high as 20 µM (see Figures 3 and 5 in Nabet et al., Nature Chemical Biology for... and we will provide an update with vendor and catalog numbers. As we are happy to share aliquots of dTAG...

...stimulate 2) Fabrication and implantation of the optical fiber into the region of interest 3) Behavioral...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one of ...10.1146/annurev-neuro-061010-113817. PMID: 21692661.  3. Bernstein JG, Boyden ES. Optogenetic tools for analyzing...Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation of which results in depolarization...optogenetics. Successful stereotaxic surgery relies on identification of the bregma and lambda sutures and subsequent...bregma which allows you to quickly and reliably locate your injection site. We recently purchased this... fluorescent protein, which allows for easy identification of opsin expression, extent of viral spread...

...which is achieved by deleting a large portion of the 3'LTR in the transfer plasmid. The deletion prevents...number of HIV genes required for virus production (3 or 4). Third-generation vectors contain gag, pol, ...lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated knockdown... looking for some additional information or clarification on these vectors. Read on to find the answers... thus decreasing the probability of creating replication-competent lentivirus (RCL) in your target cells...type or tissues; this is useful in gene therapy applications where a distinct subset of the cellular population...needs to be targeted. Q6: Are transfer plasmids replication competent? What is SIN? A6: Most (if not all)...

...PMID: 18295576. PubMed Central PMCID: PMC4142810. 3. Mitalipov, S. and D. Wolf, Totipotency, pluripotency...potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C.A....stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...Unipotency: As the name suggests, cells under this category can differentiate into only one cell type. Muscle...differentiate only into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into...somatic stem cells, these multipotent stem cells are located in small quantities in specific “stem cell niches...body. These generate specialized cells for their location, mostly to maintain cell numbers or replenish ...

...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...

...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...

...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...

...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 ...