...’t have to worry about having enough space! There’s enough space here at our new location to grow. We ...phase of the organization and the expansion of our impact, I’d like to take a moment and honor all of the... able to do good in the world. We are making an impact.  {% video_player "embed_player" overrideable=False...

...makes it easier to package for therapeutic delivery by AAV, which has strict capacity limitations. New ... RNA editing. Fast facts – PAM requirement: protospacer flanking sequence – A, U, or C. Best for: targeting...development and use. Fast facts – PAM requirement: protospacer flanking sequence – none. Best for: targeting...

...and chloramphenicol resistance pCRISPR: carries a spacer targeting the gene of interest and kanamycin resistance...template, and the locus cannot be recognized by the spacer-derived crRNA. However, if recombineering is unsuccessful...survive have inactivating mutations in the Cas9 or spacer sequence, and these rare events are easily detectable...its condensed design. For both systems, custom spacers/gRNAs are easily inserted using BbsI and Golden...proteins, and (4) the donor DNA. By using a multi-spacer CRISPR array, CRISPR-transposons can be multiplexed...

...structures, etc., as well as the presence of a Protospacer Adjacent Motif (PAM) sequence. If the gRNA sequence...and base editing applications, with high editing capacity in both contexts for NRN PAMs and to a lesser ...desired site does not have the same devastating impact on efficiency as 10-15 bps away does. This Cas ...Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361,1259-1262(2018). DOI:10.1126/science.aas9129...

...adenoviral helper genes are supplied on two different packaging plasmids: the pHelper plasmid containing Ad E2A... your infectious titer, since it will have more impact on your experimental outcome and planning.  Both... of AAV-2 particles via a novel capsid ELISA: Packaging of genomes can limit production of recombinant...resources on the Addgene blog Parts of the AAV Packaging PlasmidParts of the AAV Transfer PlasmidAAV Variables...

...may take up precious space in viral transfer vectors with limited packaging capacity. Additionally, it may...particularly when only a portion of the plasmid is packaged for viral delivery, or the relative expression...

...individual presynaptic terminals, which are jam packed with everything needed for synaptic transmission...?  ExM provides a straightforward answer to the space problem. Instead of imaging smaller, what if we ...the anchoring, digestion, and expansion steps can impact the integrity of different targets and labels. ...continuity can appear reduced in ExM, which could impact how you interpret a target’s structure. For example...

...vectors. To quantify this comparison, capsids were packed with a GFP transgene and GFP intensity and transduction...versus AAV-PHP.eB in the CNS. A GFP transgene was packaged into either an AAV-PHP.B or an AAV.PHP.eB capsid...versus AAV.PHP.S in the PNS. A GFP transgene was packaged into either an AAV.9 or an AAV.PHP.S capsid. Both...here or check out these protocols if you want to package your own virus. ...

...variable chains are secreted into the periplasmic space to be oxidized and form disulfide bonds (Frenzel...does not, be sure to set the absorbance to 280 nm (Pace et al., 1995). You’ll also need to know the extinction...Aggregation and storage Aggregation of antibodies may impact their quality and efficacy (Wang et. al., 2018)...10.1002/bit.27776. Epub 2021 May 4. PMID: 33811659. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to ...

...lost during fixation with these chemicals, which impacts sample structure and could also cause your target...destroy it for others. That said, organic solvents’ impact on lipids means that they are able to permeabilize....   Figure 1: Choice of fixation method impacts antibody binding. Formaldehyde cross-links the ...detergent has different chemical properties that will impact the level of permeabilization and membrane integrity...

... plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the journey...    The plasmid's journey begins First, each package is opened and the contents are carefully inspected...deposits arrive from labs around the globe. The packages have spent days to weeks in transit and some have...barcode can store a lot of information in a small space and is ideal for tracking large collections. Many...

...complete package, all of the pieces of software that depend on each other for the entire package to run....in-house software has been crucial for us to keep pace with our current need to analyze nearly 300 sequences...sequencing data collection and analysis eventually outpaced our software, even with continuous updates. The...

...the CNS by lentivirus, which has a larger packaging capacity than AAV. Such an approach was described ...should be screened for activity in vitro and then packaged into AAV for in vivo delivery. Multiplex gene ... also possible and is only constrained by AAV packaging limits (i.e., how many sgRNA expression cassettes...

...differing customs processes, and the distances the packages needed to travel meant the accessibility varied...such as using couriers that refill dry ice if the package stays in transit for a certain number of days and...distributor who, among other things, could ensure our packages navigated customs and arrived at their destination...for tools such as empty backbones, lentiviral packaging systems, and luciferase expression. On individual...

...lentiviral infection begins with your cells, both your packaging cells and your target cells. Poor tissue culture...harvest after transfecting with the transfer and packaging plasmids. A benefit of the multiple harvest strategy...benefitting your cells, medium exchanges may also impact the quality of the lentiviral particles you harvest...density, volume, incubation time, and MOI can all impact the success of your lentiviral infection. To ensure...

... target is present immediately upstream of a Protospacer Adjacent Motif (PAM), the sequence NGG.  If these... SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check out our SpCas9 containing...Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific modifications... within the editing window (bases 4-8 of the protospacer). While this is a small window, this system can...

...binding capacity and high protein retention. It comes in a variety of pore sizes and protein capacity, allowing...conjugate will have to be tested to ensure it doesn’t impact the antibody’s binding to your protein of interest... is a little quicker to use, with high binding capacity, though it has lower retention after binding and...10 minutes) and simple, utilizing individually packaged membrane ‘sandwiches’. They work well for proteins...

...take up essentially all of an AAV’s ~4.5 kB packaging capacity. Luckily, researchers have devised methods...the chosen acceptor vector. The vectors are then packaged into AAV virions via transient transfection of...the viral vector. This delivery method frees up space in the AAV allowing a researcher to fill it with...

...polypeptide 1 (pituitary) receptor type I PAC1, PACAPR, PACAPRI ADIPOQ adiponectin, C1Q and collagen domain...receptor 1 FLJ41949, HVR1, II, PACAP-R-2, RDC1, VAPC1, VIPR, VIRG, VPAC1, VPCAP1R VIPR2 vasoactive intestinal...cell types including T cells and B cells. It also impacts the development and function of innate immune cells...intestinal peptide receptor 2 FLJ16511, VPAC2, VPCAP2R XCL1 chemokine (C motif) ligand 1 ATAC, LPTN, LTN...

...many of the same restrictions, like limited cargo space (~4.7 kb) for your genetic payload — there are a... cell types not of interest. Systemic capsids packaging Cre-dependent transgenes can also be paired with...enhancers, and switches all take up precious cargo space in AAV vectors. Dual expression systems, which use...to be administered concurrently to minimize the impact of neutralizing antibodies developed in response...promoter/enhancer screening. However, AAV-PHP.AX packages viral genomes at a lower efficiency than AAV9 ...Zheng, Q., Kozareva, V., Beddow, T., Tobey, I. G., Pacouret, S., Chen, A. T., Chan, Y. A., Ryvkin, D., Gu,...

...recombination between homologous pairs before viral packaging even begins. This spontaneous recombination persists...2020). In general, we observe that 0.1 - 0.8% of packaged viral genomes contain a recombined/functional ...which can lead to off-target expression of the packaged transgene in neurons and glia.   Check the recombination...v14715, titer 2.4x10e13 GC/mL) for this study were packaged at Addgene and had a relatively low recombination...

...rich and diverse workforce. Out of all the career spaces I explored for my post-PhD life, the science policy...webinar via the NC Science Policy student group SciPolPack for a more detailed discussion on how to to define...to chat with someone working in a science policy space you’re interested in. Many fellowship programs can...her work focuses on incentivizing the real-world impacts of health services research. She is particularly...

...change in RNA. dPspCas13b does not require a Protospacer Flanking Sequence (PFS), making it a very flexible...editing capabilities but are small enough to be packaged in AAV particles. Want more information on the...

...CRISPR array coding for guide RNAs (diamonds) interspaced with CRISPR repeat motifs (squares), and also...concentration given relatively to the non‐targeting spacer measured by high‐throughput microscopy for a collection...

...environmental impact on a scale of 1-10: the lower the score, the lower the environmental impact in terms ... product? How can we consider the environmental impact of those processes? Who even regulates them? You...

...lentiviral capsid-based bionanoparticle system for packaging Cas9/gRNA ribonucleoproteins for transient expression...aptamer which binds an aptamer binding protein for packaging into a lentivirus-like particle. Read the paper...paper in Nucleic Acids Research Find the gRNA and packaging plasmids   Cytosine deaminase for RNA editing...

...this time more visible and thereby have a positive impact on science. ReFigure saves time, knowledge and ...definitely give you credit for it and increase your impact! Download ReFigure extension and visit ReFigure.org... with Joe Akin. She envisions a world where the impact of teaching and doing science is rapid, concrete...

...the focus shifts to choosing an unexplored and impactful research topic that aligns with your interests...when selecting a research project. What will the impact be in a best case scenario? If you get your dream...what will your paper look like? What will be the impact on your field? You want to choose a research question...

..."CRISPR", short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned...want to use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The...usually adds it when they order the oligo. If your spacer sequence starts with a 'G', you naturally have ...

...immune system, CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) pathway, as an ...matching region on the sgRNA and a short DNA motif (protospacer adjacent motif or PAM, sequence: NGG) juxtaposed...