...without double strand breaks (DSBs) or donor DNA templates. How does this work? First, an engineered prime...reverse transcriptase domain uses the pegRNA to template reverse transcription of the desired edit, directly...increase activity, enhance binding between the template and primer binding site, increase processivity...different genomic sites compared to PE1. The pegRNA: A template and guide all in one The other important component...pegRNA is a guide RNA that also encodes the RT template, which includes the desired edit and homology ...remake that strand using the edited strand as the template. A third prime editing system called PE3 does ...increase thermostability through tighter binding to template-primer." Nucleic acids research 37.2 (2008): 473...

... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...making the cut off essentially as part of the templated DNA. Check out our blog post on increasing HDR...To make sure your donor molecule is used as the template, you need to incorporate homologous sequence to...sister chromatid is likely to exist as a repair template. To maximize HDR events, ensure cells are actively...

...one AAV and a repair template in second a second AAV to deliver their repair template to mouse zygotes and...homologous recombination with a homologous repair template. It is this application - the creation of sequence-specific...allowing a researcher to fill it with the repair template. Bok and Porteus 2017 recently showed that by ... as RNPs and delivering two halves of a repair template spread across two AAVs, it is possible sequentially...this technique, Bok and Porteus deliver a repair template that would take up ~6.5 kB if delivered in a single...technique to efficiently deliver large repair templates for applications that require large gene fusions...

...with ssDNA repair templates. Their work demonstrates that different repair template strategies activate...using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template to increase ...working with CRISPR than just designing a repair template that contains your desired edit, and I want to...Editing in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and ...

...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...the second site accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner... and repressors, can be included in the repair template for inducible alteration of protein expression...

... science.                    Two of the platforms we use at Addgene: YouTube, for longer educational...octogenarians is going to be a hard sell!) Digital platforms often have demographic data of their users readily...geographic location. For example, not all social media platforms are available or used in all countries; not all...the limitations and potential of your preferred platform before you start can save a lot of trouble in ...requirements.  No visuals. Variety of hosting platforms can splinter audience. Primarily digital distribution...communicators. Well-developed content creation platforms.  Algorithms can make it difficult to reach people...

... to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar...point, they hope that the protocol serves as a platform for advancing diagnostics.     DETECTR- DNA...harnessed this ability to create a DNA detection platform called DETECTR. How does DETECTR work? DETECTR...technology with the mission to “provide a CRISPR-based platform on which an infinite number of tests” for biosensing...Biosciences and UCSF recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip...Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360:439–...

...promoter or the coding sequence and the template or non-template strand, among other parameters. .  Stanley...introduced along with an oligonucleotide repair template. Through recombineering, the locus of interest...interest is modified to match the repair template, and the locus cannot be recognized by the spacer-derived ... contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Each round of...with XbaI to insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning... WA (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression...

...assembly cloning platform called GMAP (Gibson assembly-based modular assembly platform). GMAP is based ...orphan GPCRs demonstrating the utility of this platform for drug discovery. Researchers can use plasmids...Kit are available as glycerol stocks in a single plate with every plasmid or as individual plasmids shipped...Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of which greatly enhances the.... PubMed PMID: 26165496 GMAP - A new assembly platform for generating both simple and complex DNA constructs...capabilities of Gibson assembly and make a more modular platform, the Jacks lab generated 5 common 30bp overlap...However, the laborious construction of repair templates and guide RNA constructs, as well as the difficult...

...antibiotic resistance marker. After transformation, plate the cells on medium containing the corresponding...contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing agar. Only cells... are auxotrophic for specific amino acids. When plated on media lacking the essential amino acid, the ... a toxic metabolite in gram-negative bacteria. Plating on sucrose medium will select for cells that contain...gene is used for negative selection on 5% sucrose plates. Exchange of the sacB gene for the gene of interest...

...DNA from each well of a 96-well plate. Then, samples from several plates are pooled together to create ...samples individually, plexWell™ reduces every 96 well plate to a single tube after the first tagging step. As... samples (e.g. a collection of 96 plasmids on a plate) will typically have a wide range of input quantities... from plexWell™ NGS data. Once Addgene produces plates of plasmid DNA, they are shipped to seqWell, where...

...devices for research. There are a number of new platforms (such as openUC2, which I am a co-founder of),...that make everyday microscopy much easier. On platforms such as Github, users can download the software...example, can also be printed quickly in Madagascar. Platforms that allow for on-going creativity with the construction...organizations and companies to provide problem-solving platforms.     Fig. 3: Different workflows involved...Prakash, "Squid: Simplifying Quantitative Imaging Platform Development and Deployment." bioRxiv. 2020. Diederich...

...engineers regularly use the platform, why scientists are using the platform, and tips for getting the most...from Nature Jobs Blog While Twitter is a popular platform for scientists, it’s not the only tool in a scientist... Facebook. For each of the three social media platforms, there’s some statistics on what percent of scientists...given you tips for using various social media platforms, and 3) helped you fit social media into your ...

..., and electroporated them with an ssODN repair template in induced pluripotent stem cells made from the...fertilization. Since Ma et al. designed a repair template with two synonymous mutations to distinguish it...were able to distinguish between repair from the template vs repair from the maternal MYBPC3 allele. Interestingly...from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but instead from the mother...embryos to undergo editing using the provided ssODN template. In fact, this finding led the authors to title...

...secondary nanobodies blog post.  A rapid selection platform for generating sybodies against membrane proteins...weeks (Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly...in the form of mRNA for academic research. The template plasmids for generating your own libraries are... out with anti-Myc antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated...proteins. The full protocol for this sybody selection platform can be found here and relevant plasmids are available...

...shaking at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase ...to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted...Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic... and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming ...Molecular Biology Protocols Over-Agar Antibiotic Plating Plasmids 101 eBook Resources on the Addgene blog...

...then recognizes the encapsidated minigenome as a template for replication and transcription, as it would...-minigenome). The anti-minigenome is used as a template by the viral polymerase to synthesize additional...addition, the polymerase will use the minigenome as a template to transcribe the reporter gene (e.g. firefly ...polymerase complex and utilize the minigenome as a template. Thus, it is important to choose cell lines that...and 2 μg minigenome plasmid per well (in a 6 well plate format). However, since plasmid quality and transfection...

...genome-wide CRISPR inhibition Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference...inhibition library Covalent tethering of DNA repair template to Cas9 Homology-directed repair (HDR) can be ...using a single-stranded oligodeoxynucleotide HDR template covalently attached to the Cas9-gRNA RNP complex...Candida albicans A new CRISPR interference (CRIPSRi) platform for targeted gene repression in Candida albicans...

...created a bacterial lawn on an agar plate, put a mask over the plate containing the pattern of the image...image, and shined blue light over the plate. Areas on the plate that are blocked by the mask will not produce...

...establish a next-generation sequencing (NGS) QC platform that allows us to provide scientists with complete...launching our viral service in 2016, we adapted this platform to accommodate adeno-associated viral vector (...ligation and tagmentation require a double-stranded template.  Using viral genome sequencing to verify the ...double-stranded species that could be used as a template for downstream applications (Berns, 1972). Using...