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We narrowed to 573 results for: BASE

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  1. FPbase: A new community-editable fluorescent protein database

    Type
    Blog Post
    Published
    May 16, 2018, 1 p.m.
    ...best” FPs, and I collaborated with him on a web-based interactive visualization of FP attributes (which...about FPs. FPbase.org FPbase is a project written in Python & Django, using a popular database management... of information in the database. Plans for future development of FPbase FPbase is still very much in the...community-editable database is free from the risk of inaccurate data submissions, so FPbase includes a versioning...possible after submission. Using FPbase The most obvious use case for FPbase is finding information on a particular.... The need for a community fluorescent protein database As recognized by the 2008 Nobel Prize, fluorescent...additional references, etc...) and a more robust database structure was needed.  It is also difficult to...

  2. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    Published
    Feb. 13, 2025, 2:15 p.m.
    ...out of 12 possible base-to-base changes. The other 8 base-to-base changes require Base Transition Editors...correct single-base mutations without introducing DSBs at all. Base editing to the rescue! Base editors chemically...first two classes of base editors were cytosine base editors (CBEs) and adenine base editors (ABEs). CBEs...Both are base transition editors: converting one purine base to the other or one pyrimidine base to the ... prevents base excision repair, which would change the U back to a C base. To increase base editing efficiency...Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base changes. ...following base editing and DNA repair. Black arrows indicate natural cellular processes. BER = base excision...

  3. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    Published
    April 1, 2025, 1:15 p.m.
    ...unmodified G bases, resulting in a G → Y (Y = T or C) base editor, which they called glycosylase-based guanine... in the base editing field! From CRISPR-free approaches to mitochondrial base editing to base editors ...   Base transition Base change between purines (A ↔ G) or between pyrimidines (C ↔ T).   Base transversion...type of base editor invented. ABE Adenine Base Editor Converts A → G; the second type of base editor ...C), X (any base), or C, respectively. gGBE, gTBE, gCBE Glycosylase-based G, T, or C base editors Excises... we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs first convert...this post! Figure 1: Base transitions are edits between the purine bases (adenine, guanine, and hypoxanthine...

  4. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    Published
    Jan. 3, 2019, 1:35 p.m.
    ...applied to fix the defective base precisely? Until 2016, precise single base changes were only possible ...CRISPR-mediated base editing (BE), these types of repairs can now be done more efficiently than before. A base editor...plant genome editing. Cytosine base editors (CBE) Precise and efficient base rewriting in the genome became...T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation by base editors...in CBE. Base editors for the plant genome These preliminary studies demonstrated successful base editing...adapting and improving base editing platforms for plant systems.  Figure 2: Plant base editor platforms...components. Adenine base editors for plant We developed plant adenine base editor (ABE) vectors to...

  5. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...first base editors revolutionized CRISPR gene editing. Cytosine base editors (CBEs) and adenine base editors...out of the possible 12 base-to-base conversions. The other 8 conversions are base transversions: they require...cellular process to repair DSBs. Base Editing terms   Base transition Base changes between purines (A ↔ ...Cytosine Base Editor Converts C → T; the first type of base editor invented. ABE Adenine Base Editor ... of base editor invented. CGBE C → G Base Editor Converts C → G; also called a glycosylase base editor...first base editors were both deaminases: they removed an —NH2 (amine) group from the target base and replaced...conversion between the purine bases (A and G) or between the pyrimidine bases (C and T). (Check out our previous...

  6. Expanding the Targeting Scope and Editing Efficiency of Adenine Base Editors

    Type
    Blog Post
    Published
    March 17, 2020, 1:32 p.m.
    ...deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from... precision editing capabilities. Base editors make specific DNA base changes and consist of a catalytically...deaminase induces the DNA base change without a DNA double-strand break. But base editors aren’t perfect....sites, or make only a subset of base substitutions.  The need for speed: base editing before Cas lets go ...issue that arises for base editors is that the editing domain must induce the base change before the Cas.... A plasmid-based genetic circuit in the bacteria links the expression of gene III to base editor activity... 2: Phage-assisted evolution of base-editing activity. A plasmid-based genetic circuit in the bacteria...

  7. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    Published
    July 7, 2020, 1:15 p.m.
    ... only easily identify base editing events in real-time but also enrich for base-edited cells in their ... real-time, fluorescent based identification system for the isolation of base-edited cell populations.... the Dow lab created a base editing reporter that detects and enriches for base editing events in vivo...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...past few years, scientists have created an array of base editing reporters that can help you do just that...create the inactive mCherry, the lab introduced a 43 base-pair insertion that disrupts fluorescence due to...trinucleotide motifs (5’-TCA-to-TUA) found within the 43 base-pair mCherry insertion. Editing of these motifs ...

  8. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    Published
    May 7, 2020, 1:15 p.m.
    ... targeting scope for base editing.  Try ABE8 base editors for your research! Base editing in primary human...Adenine base editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make... maintaining on-target base editing functionality.  The delivery method of base editors plays a role for... Find more blog posts about base editing Learn about another evolved base editor: ABE8e Read about xCas9... make these base changes, especially in primary human cells. Now, scientists at Beam Therapeutics have... (Gaudelli et al., 2020). One of the widely used base editing systems, ABE7.10 (and the starting point..., 2020. Directed evolution identifies adenine base editors with improved editing in cell lines Nicole...

  9. High Complexity Golden Gate Assembly with NEB’s DAD

    Type
    Blog Post
    Published
    Aug. 1, 2023, 1:15 p.m.
    ...°C 1 hour 3-base or 4-base T4 DNA ligase 25 °C 18 hours 3-base or 4-base T4 DNA ligase...°C 1 hour 3-base or 4-base T4 DNA ligase 37 °C 18 hours 3-base or 4-base Bbsl-HF®, 1x... 4-base Bsal-HFv2 37-16 °C cycling 4-base BsmBI-v2 42-16 °C cycling 4-base BsmBl-v2...used. Each of the tools works with either 3-base or 4-base overhangs, and with a variety of different ...MM 37-16 °C cycling 4-base Bsal-HFv2® 37 °C Static 4-base Bsal-HFv2, 1x NEBridge Ligase...°C cycling 4-base BspQI, 1x NEBridge Ligase MM 42-16 °C cycling 3-base  PaqCl®, 1x T4...°C cycling 4-base PaqCl, 1x NEBridge Ligase MM 37-16 °C cycling 4-base Sapl              ...

  10. What's New in CRISPR - December 2019

    Type
    Blog Post
    Published
    Dec. 17, 2019, 2:37 p.m.
    ...in Burkholderia Base editors CRISPR toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein...SECURE adenine base editor Earlier this year, Keith Joung’s lab developed SECURE cytosine base editors, which...paper in Nature Biotechnology Find the base editors   Single base editor that induces C-to-T and A-to-G...recently developed a base editor that combines the abilities of C-to-T and A-to-G base editors into one protein...Cas9-based toolkit for epitope tagging in neurons Harold MacGillavry’s lab developed a CRISPR-based toolkit... APOBEC1. Now, they’ve engineered SECURE adenine base editor variants using a protein-truncation strategy...efficiency of a target site compared to a mixture of two base editor enzymes in mammalian cells. Read the paper...

  11. DAP arrays for multiplex gene editing

    Type
    Blog Post
    Published
    July 7, 2022, 1:15 p.m.
    ...Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored for efficient base editing. ...possible to use the state-of-art base editor, prime editor, or any CRISPR-based gene editors, for correcting...strategies have been facing difficulties in pairing with base editing and prime editing approaches. Recently, ...has developed DAP arrays for efficient multiplex base editing (MBE) and multiplex prime editing (MPE) ...regulations, it lacks proper functions to enable efficient base editing and has yet to be developed for prime editing...CRISPR-Cas9, the key component to enable efficient base editing and prime editing, is unable to process ...array but is the key component to enable efficient base editing and prime editing. How do you then set up...

  12. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    Published
    March 6, 2018, 1:49 p.m.
    ... for proper base pairing through minor grove contacts, and thus do not read specific base sequences, but...hydrogen bonds and a base-pair size nearly indistinguishable from a Watson-Crick base pair, are joined with...the base pair providing the 3 ́-OH terminus to the ligation) than on the downstream side (the base pair...each differing only in the base at the ligation junction. Thus, all four bases at either side of the ligation...structures, including substrates containing gaps or DNA base pair mismatches (8-11). For some applications, there...there is a need for the exclusive ligation of fully base-paired nicks with no gaps. For example, DNA assembly... suitable probes, LDR can reliably detect single-base differences. The original creators of this technique...

  13. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    Published
    March 21, 2019, 12:47 p.m.
    ...plasmids containing these base editors Spatially and temporally regulated AAV-based genome editing In 2016... plasmids CRISPR-based method for cell lineage tracing Valcyrie is a CRISPR-based marker excision method...Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion Caixia... Caixia Gao’s lab has developed a new plant base editor from an evolved tRNA adenosine deaminase that ...that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits in protoplasts or plants...the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated not only temporally...Frontiers in Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly...

  14. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    Published
    Oct. 13, 2022, 1:45 p.m.
    ...without undesired bases nearby that may undergo unwanted editing. Just like with base editing, alternative...modified to enable researchers to knock out, knock in, base edit, activate, repress, and even image your favorite...Requirement and Expanding CRISPR Beyond SpCas9.” Single base pair edits Is the location and type of edit you ... your precise edit without even making a break.  Base editors are dead Cas9 (dCas9) or nCas9 enzymes fused...cytosine or adenosine deaminases to facilitate a single base pair edit. (Cytosine deaminases edit C-to-T and ... the programmed edit within the editing window (bases 4-8 of the protospacer). While this is a small window... desired edit will be performed on all relevant bases within the editing window, for example all C’s will...

  15. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    Published
    Aug. 27, 2015, 2:30 p.m.
    ... used with a wide variety of restriction enzyme-based cloning methods. Each method has its own pluses ...outside of their recognition sequence, creating four base flanking overhangs. Since these overhangs are not... original plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden ...Golden Gate cloning is its scalability. Unique 4 base overhangs can be used to assemble multiple fragments...advantages over other cloning methods. Exonuclease-based methods like Gibson assembly require 20-40 bp of...DNA fragments at a time. As with all Golden Gate-based methods, this system exploits the ability of Type...Bogdanove and Voytas groups described a new Golden Gate-based technology for genome editing which allowed for ...

  16. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    Published
    March 3, 2020, 2:15 p.m.
    ...therapeutic potential for the split base editors. Find the split base editors at Addgene. Levy et al., Nature...please sign up here.        AAV delivery of split base editors Article contributed by Jennifer Tsang AAVs...limits the delivery of larger transgenes, such as base editors which have a gene size of ~5.2 kb. To overcome...David Liu’s lab developed split cytosine and adenine base editors that can be reconstituted in vivo to form...sequence to the unmodified enzyme. Each half of the base editor is fused to a fast-splicing split intein ...peptide bond at the split site. Figure 1: The base editor is split into two plasmids and co-transfected... a single AAV expressing GFP. The lab tested the base editors in vivo and achieved therapeutically relevant...

  17. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    Published
    April 11, 2017, 2:30 p.m.
    ...applications that lend themselves to aptamer-based instead of protein-based fluorophores, including: Environments...course of the next two decades, these nucleotide-based ligand binders would prove to be highly adaptable... few important advantages over their amino acid-based counterparts, antibodies and enzymes, such as: ...many entirely new applications including aptamer based fluorescent biosensors. Aptabeacons Fluorescence...instance, the fluorophore and quencher may form the base of the stem in an RNA stem-loop structure (see Fig...compound malachite green (MG) to create a non-protein based visualization technique. Ultimately, MG proved to...eukaryotes, the Jaffrey lab has since developed new RNA-based scaffolds to contain and stabilize Broccoli cassettes...

  18. Design Tips for Prime Editing

    Type
    Blog Post
    Published
    Jan. 23, 2025, 2:15 p.m.
    ...The first base of the 3′ extension of the pegRNA should not be C. A C base is speculated to base pair with...begins with a C base can cause unwanted base pairing (left), while pegRNAs that use other bases in this location...near point mutations to create 3-base (or longer) tracts of edited bases. DNA mismatch repair (MMR) is less...efficiently targets single-base mismatches (left), but “bubbles” of 3 or more mismatched bases can often evade MMR...efficient at identifying these “bubbles” of mismatched bases, enhancing correct editing efficiencies (Chen et... create linkers that minimize unwanted intra-RNA base pairing with the primer binding site (Nelson et ...

  19. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ... than base editing (in certain circumstances) Current base editors can perform any base-to-base conversion...12 possible base-to-base changes in a single edit. Prime editing is also more precise. Base editors, for...targeted insertions, deletions, and all possible base-to-base conversions (Anzalone et al., 2019). Plus, it...can be over 30 base pairs for prime editing. Since PAM sites occur on average every ~8 base pairs on either...DNA strand, many previously developed base editors with a <8 base-pair editing window cannot edit within... example, typically edit all the target bases within the base editing window, while prime editors make...nucleotides are positioned within the canonical base editing window, base editing has higher efficiency and fewer...

  20. Visualizing Genomic Loci with CRISPR-Sirius

    Type
    Blog Post
    Published
    April 18, 2019, 12:53 p.m.
    ...widely adapted for a wide variety of applications: base editing, CRISPR activation and inhibition, among...visualization tools is CRISPR-Sirius, the brightest CRISPR-based tool to date for visualizing genomic loci in living... Massachusetts Medical School developed a dCas9-based multicolor labeling system to visualize chromosomal...St1Cas9. These fusions localized to specific loci based on the guide RNA sequence. A year later, they developed...et al., 2018). They improved on previous CRISPR-based loci visualization tools by increasing gRNA stability... aptamer insertion in the gRNA tetraloop (a four base hairpin loop motif within the gRNA) resulted in ...loci gave a detectable signal in human U2OS cells. Based upon this, they chose an intergenic DNA region (...

  21. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    Published
    May 27, 2025, 1:15 p.m.
    ...which kit to use for your experiment. Bead-based or column-based kits — which to choose? Plasmid purification... column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications...illustrates the three grades of DNA you may need based on their relative quality and quantity.   ...1: Plasmid preps fall into different categories based on their relative qualities and quantities. Image...eluting the DNA from the substrate. In a column-based approach, the substrate is a silica matrix fixed... usually by centrifugation or vacuum. In a bead-based approach, you mix specially coated magnetic beads...ultimate plasmid yield.  Take your average column-based miniprep kit for example. These kits are designed...

  22. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ...the black plague).  Base editors The major drawback of all the endonuclease-based gene editors (meganucleases...enzyme to generate the prototypical base editor enzymes. The cytidine base editors (CBE) use a cytidine deaminase...understanding of the adenine base editors which can help accelerate the engineering of novel base editor proteins....movie’s name is itself a combination of the four bases of DNA – A, T, G, and C. In this movie Ethan Hawke...several ZFNs are strung together, it can read many base pairs simultaneously to increase specificity. This...as CRISPR-Cas editing soon replaced the protein-based DNA-binding domain with a single-guide RNA sequence...the genome. This was soon followed by the adenine base editor (ABE) enzyme in 2017. ABEs rely on a similar...

  23. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    Published
    March 27, 2018, 1:32 p.m.
    ...CRISPR-based approaches, several groups have recently worked to enhance the efficiency of AAV-based gene...gene is removed via infection with adeno-cre. AAV-based gene editing compared to CRISPR editing CRISPR, ...more challenging to develop high efficiency CRISPR-based approaches for the generation of knockins via homologous...However, there are important disadvantages to the AAV-based approach that must be considered when deciding whether...experimental approach. First, unlike CRISPR, the AAV-based approach is only able to target a single allele ...the same cell, are more appropriate for a CRISPR-based approach. Second, the AAV preps generally used for...in a given experiment. For this reason, the AAV-based approach requires G418 selection of infected clones...

  24. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

    Type
    Blog Post
    Published
    Sept. 18, 2018, 1:33 p.m.
    ...  Listen to the base editing podcast segment The increasing popularity of single base-editors has resulted...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to ...imaging capabilities. Upon developing a new imaging-based bacterial colony screening platform, Oliver Griesbeck...fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity Article contributed...editing toolkit. However, a challenge in optimizing base editing technologies across several cell types is... human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more efficient than the BE3...

  25. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
    Published
    July 21, 2020, 1:15 p.m.
    ...generation sequencing. Unlike absorbance-based methods, fluorescence-based methods require a standard curve, ...contamination based on the presence of other bands or streaking. Unlike absorbance based methods, this...ways to do this and the method you choose could be based on your downstream application, time, and instrument...correct size. This is something that absorbance based methods can’t tell you and you don’t need a spectrophotometer...of your DNA at different dilutions and quantify based on band intensities of your DNA relative to the ...plasmid prep. Diphenylamine method Another absorbance based method for quantifying DNA uses diphenylamine. Diphenylamine...

  26. Plasmid Cloning by PCR

    Type
    Blog Post
    Published
    March 29, 2016, 2:30 p.m.
    ... You can generally add any 6 bases, but you should ensure that the bases do not result in the formation...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time...a recipient plasmid. Designing primers for PCR based cloning     The basic PCR primers for molecular...molecular cloning consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction...information). Thus, we recommend that you add 3-6 bases upstream of your restriction site to improve cutting...amplification. We can start similarly, taking the final 18 bases of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA... is. You should select an annealing temperature based on the melting temperature (Tm) of the portion of...

  27. Even more elegant: Single injection CRISPR/Cas9 in C. elegans

    Type
    Blog Post
    Published
    July 7, 2015, 3:36 p.m.
    ...of hands-on time. Creating a modular, selection-based gene tagging approach Multiple features of C. elegans... the selection marker. Other labs have used PCR-based screening to detect mutants, but this method limits... sought to create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins on any ...Genetics Society of America. To use this SEC-based system, first design and clone 500-700 bp homology... candidate animals can then be further screened based on their Roller phenotype. If SEC is inserted at...animals that have undergone SEC excision are screened based on the loss of the Roller phenotype. These animals...attempt for 6/7 genes. The data are clear - the SEC-based system is both simple to use and highly efficient...

  28. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    Published
    June 4, 2024, 1:15 p.m.
    ...’, where N can be any base. In Figure 5, we can see why: two arginines make base-specific hydrogen bonds...stability for the next DNA base pair to be unwound. A mismatch within the 10-base seed sequence will destabilize...guide RNA and wondered why it is limited to only 20 bases, or why it’s so important to choose a target sequence...nucleotides 1-10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10...only bound by the phosphate backbone. These PAM bases must be present for Cas9 to unwind the DNA and check...Residues R1333 and R1335 from the CTD (yellow) make base-specific hydrogen bonds to guanines of the PAM in...complementarity with its guide RNA. But how to test DNA bases that are already snugly paired to each other? At...

  29. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...the content of our paper, “Synthetic recombinase-based state machines in living cells” (1), into a more...how to adapt our engineered devices, recombinase-based state machines for your own experimental needs. ...the order or timing of these factors. Recombinase-based state machines (RSMs), which take on a particular...particular DNA-sequence configuration (state) based on the identity and order of a particular set of inputs, ...Sanger sequencing, qPCR, and fluorescent reporter-based) each come with their advantages and disadvantages... the predominant state of registers in a sample based on a similarity metric that we defined in our paper...convenient strategy is the fluorescent reporter based method since it lends itself to cheap, quick, and...

  30. QC Sequencing Technologies at Addgene

    Type
    Blog Post
    Published
    July 15, 2025, 1:15 p.m.
    ...instrument changes. Base-calling software can then identify the nucleotide that passed through based on these characteristic... relies on “sequencing-by-synthesis,” where each base is recognized and recorded as a DNA strand is synthesized... reactions, where a fraction of one of the four bases (A, C, T, or G) is supplied as a dideoxyribonucleoside...sequencing results are deconvoluted to identify the bases (and, if necessary, separate different samples),...and Sanger sequencing are limited to hundreds of base pairs, long-read sequencing can read sequence lengths...lengths in the thousands or even millions of base pairs. Several companies have worked on this technology...Nanopore sequencing deviates from the synthesis-based sequencing methods (Figure 3). It uses (aptly named...

  31. Hot Plasmids and Viral Preps - July 2021

    Type
    Blog Post
    Published
    July 27, 2021, 1:15 p.m.
    ...Here’s what you’ll find in this article: CRISPR-based editing in organoid Biosensors for detecting strigolactones...ready-to-use viral preps New toolkit for CRISPR-based genome editing in organoids By Ashley Waldron Organoids...organoid users have recently begun to develop CRISPR-based approaches for studying gene function in these systems...other tissue-types. By bringing together CRISPR-based approaches and organoid models, toolkits like this... SLs. The authors also showed that the receptor-based fluorescent biosensor has a high sensitivity (50...and validate a ratiometric fluorescent biosensor based on the parasitic Striga hermonthica SL receptor ... Design and mechanism of action of the receptor-based SL fluorescent biosensor. Image from Chesterfield...

  32. MXS Chaining

    Type
    Blog Post
    Published
    Feb. 7, 2017, 3:30 p.m.
    ...MXS-chaining is repeatable, restriction-enzyme based (chaining-based) cloning. The modules (Figure 2) are contained...BioBricks, Bglbricks, etc. Chaining-based methods (restriction enzyme-based). Well suited for the modular ...methods are listed in Table 2. For example,  chaining-based cloning methods require that the separate modules...2008; Anderson et al., 2010 Among the chaining-based methods, the specific restriction enzymes used affect...specific restriction enzymes used in various chaining-based cloning methods are listed in Table 3. The goal ...emailing us at [email protected]. Table 3: Chaining Based Cloning Methods Method Restriction Enzymes ...deoxyribonucleic acid. VIII. Frequencies of nearest neighbor base sequences in deoxyribonucleic acid. The Journal ...

  33. Hot Plasmids - August 2020

    Type
    Blog Post
    Published
    Aug. 18, 2020, 1:15 p.m.
    ....  Here's what you'll find in this post: Yeast-based protein evolution Fluorescent tools for mammalian...the viral service Faster catalysis after yeast-based protein evolution By Joanne Kamens Mateo Sanchez...optimize another. The authors have created a yeast-based directed evolution approach with some notable improvements...antiviral CRISPR in human cells) is a CRISPR-Cas13 based antiviral method to degrade RNA from SARS-CoV-2 ... 95 plasmids consisting of CRISPR/Cas nucleases, base editors, gRNA backbones, and promoters for expression... the plasmids at Addgene! A new dual-deaminase base editor enables concurrent adenine and cytosine editing...control gene expression. This includes a CRISPRi-based synthetic oscillator, bistable network, stripe pattern-forming...

  34. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    Published
    Dec. 14, 2021, 2:15 p.m.
    ...restrictions, anything within 5 base pairs should work well, and up to 10 base pairs away (especially 3’ of...introduce you to a simpler, yet more effective, CRISPR-based epigenetic approach (spoiler alert: it’s dCas9… ...When I started to work on building a CRISPR/dCas9-based tool for DNA demethylation in 2015, none existed...that oxidize 5-methylcytosine to create rare DNA bases that are recognized by distinct proteins and result...Kaas et al. 2013). Figure 2: CRISPR TET-based epigenetic editing. In reality, editing your target...system to balance these needs is the lentiviral-based expression of floxed dCas9 so that it may later ...at McGill University, working to develop CRISPR-based tools for epigenetic editing.   References and resources...

  35. Adapting Toehold Switches to Detect Zika Virus

    Type
    Blog Post
    Published
    Aug. 30, 2016, 2:30 p.m.
    ... et al, 2014). The second was an in vitro paper-based platform that allowed gene circuits to be deployed... of these combined technologies, we built paper-based synthetic gene networks for the detection of antibiotic...work was exciting because it enabled gene circuit-based sensors to be used outside of the laboratory. The...the lab had been working on advancing the paper-based platform toward a more field-ready format when the...that, given the critical need for low-cost, field-based diagnostics, our approach could make an impact. ...paper when we say the timeline to develop our paper-based platform into clinically relevant diagnostics for...week (Pardee et al, 2016). Using toehold switch-based RNA sensors, our approach uses portions of the pathogen...

  36. FlipGFP, a novel fluorescence protease reporter to study apoptosis

    Type
    Blog Post
    Published
    May 21, 2019, 12:10 p.m.
    ...as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In these reporters...in fluorescence emission. FRET and fluorescence based reporters however have several limitations. First...also designed a red fluorogenic protease reporter based on superfolder Cherry (sfCherry). Like FlipGFP, ...used to cleave proteins. This protease reporter based on sfCherry however did not initially fluoresce ...). Studying apoptosis in vivo using the FlipGFP-based caspase reporter The Shu Lab successfully used FlipGFP...Lastly, the Shu Lab demonstrated that the FlipGFP-based caspase reporter can be used to visualize apoptosis...et al. "A new protein-protein interaction sensor based on tripartite split-GFP association." Scientific...

  37. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    Published
    April 23, 2020, 1:15 p.m.
    ...studies used SMRT sequencing, TA-based ligation and tagmentation-based methods for AAV genome sequencing...only works for self-complementary AAVs and the TA-based ligation and tagmentation preparation approaches...because the AAV genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded...Berns, 1972). Using qPCR and restriction enzyme based methods, we showed that the DNA extract is a heterogeneous... serve as effective substrates for tagmentation-based library preparation procedures. This finding eliminates...data. Unlike previously described thermodynamics-based methods of serotype confirmation, this method does...

  38. Hot Plasmids and Viral Preps - September 2021

    Type
    Blog Post
    Published
    Sept. 21, 2021, 1:15 p.m.
    ...ABE’s. b) Quantification of base-editing efficiency using XMAS-TREE-based methods at various genomic loci...enrichment of ABE-targeted cells By Ellie Adams Single base modifications through ABE or CBE's have the potential... deposited a set of plasmids that helps identify base edited cell populations without end-point sequencing...for editing enrichment”, or XMAS-TREE, to identify base edited cell populations without end-point sequencing...library targets E-boxes genome-wide. It was designed based on MYC-ChIP-seq data from several MYC-dependent .... The BARBEKO sgRNA library uses CRISPR cytosine base editors to disrupt gene function by targeting start...

  39. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    Published
    April 7, 2015, 4:21 p.m.
    ...publication: http://www.freebase.com/base/aptamer Cruz-Toledo, J. et al. Aptamer base: a collaborative ...collaborative knowledge base to describe aptamers and SELEX experiments. Database, bas006 (2012). PubMed. For a ...biofactories by demonstrating the use of CRISPR-based RNA scaffolds to mimic natural transcriptional programs... RNA scaffolds follows the rules of Watson-Crick base pairing, allowing for rational, straightforward ...straightforward design. Since the technique relies on base pairing, it has promise to be host-independent, beyond the...the University of Western Ontario studying plant-based recombinant pharmaceuticals. Follow him on Twitter...

  40. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ...utility of cytidine and adenine base editors that enable C-to-T and A-to-G base substitutions respectively.... were first discovered in bacteria, most CRISPR-based genome engineering has taken place in other organisms...CRMAGE system combines CRISPR and recombineering-based MAGE The Nielsen lab’s CRMAGE system is a fast, ... method that combines CRISPR and recombineering-based MAGE (Multiplex Automated Genome Engineering) technology...Bikard et al., 2013), the Qi lab employed a gRNA-based design more familiar to those using CRISPR in eukaryotes...catalytically dead Cas9 enzyme allows reversible repression based on the presence of the inducer, in this case anhydro-tetracycline...RNAP-Omega-dCas9 fusions. Here’s some others. SoxS based CRISPRa In the search for a CRISPR activation method...

  41. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    Published
    March 28, 2018, 6:52 p.m.
    ...IDT estimates that there is an NGG PAM every 42 bases in the human genome. SpCas9 targeting should be ...difficult if there’s no PAM near your insertion site. Base editing, another method to create point mutations...Employing xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional...NGG PAM, confirming the flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible...nuclease, xCas9 3.7 fused to third generation cytidine base editor architecture (BE3) slightly improves NGG ...efficiency over that of SpCas9-BE3 (37 vs 28%). Cytidine base editing at NGT, NGC, GAA, and GAT PAMs is improved... with a smaller increase in NGA editing. Adenine base editor xCas9 3.7-ABE also follows the same pattern...

  42. New CRISPR Web Resources and #12DaysofCRISPR Recap

    Type
    Blog Post
    Published
    Dec. 12, 2017, 3:13 p.m.
    ...that cover some of the hottest CRISPR tools. Base Edit: Base editing plasmids allow you to make precise ...causing double-strand breaks. Our new page shows you base editing plasmids for mammalian systems, bacteria...bacteria, plants, and yeast. Popular cytosine base editor pCMV-BE3 has been requested nearly 400 times, and ... has already earned a blue flame for its adenine base editing capabilities. Epigenetics: CRISPR has made...for Gene Editing Day 9: Precision editing: Single Base Editing with CRISPR and Enhancing CRISPR Targeting...

  43. IBEX Knowledge-Base: A Data Resource for Multiplex Tissue Imaging

    Type
    Blog Post
    Published
    June 8, 2023, 1:15 p.m.
    ...IBEX Knowledge-Base, with every contributor listed as an author.  The IBEX Knowledge-Base is an invaluable...amplified staining methods, and oligonucleotide-based staining. Of course, if you’re interested in using... Ziv Yaniv, PhD, have created the IBEX Knowledge-Base, a community repository and resource for spatial...open-software community when designing the IBEX Knowledge-Base, seeking to create an iterative process that is .... Everyone who contributes to the IBEX Knowledge-Base is named on the Zenodo release in keeping with their...components of building both the community and the database. They particularly encourage people to upload ...

  44. What's New in CRISPR - March 2020

    Type
    Blog Post
    Published
    March 10, 2020, 1:15 p.m.
    ...plasmids Cytosine base editors with minimized off-target DNA editing The original cytosine base editor, BE3,...research! This time: Optogenetic repression in yeast Base editing CRISPRi in Caulobacter crescentus Multiplexed...Multiplexed gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene... Read the paper in Nature Biotechnology Find the base editor plasmids CRISPRi in Caulobacter crescentus...Scientific Reports Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components...

  45. The Twelve Days of CRISPR

    Type
    Blog Post
    Published
    Dec. 22, 2020, 2:15 p.m.
    ... on base editing from the first generation of base editors to the eighth generation (and dual base editors...Addgene gave to me: SHERLOCK and DETECTR, two CRISPR-based methods to detect nucleic acids.  On the third day...

  46. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    Published
    May 8, 2018, 1 p.m.
    ...in the region around the cut site. It shows, base-by-base, the average amount of signal that disagrees...Editing Results with ICE on Synthego’s blog. CRISPR-based genome engineering has revolutionized the gene editing...Sanger sequencing data alongside the analysis of NGS-based amplicon sequencing data and found that the accuracy...enabling a ~100-fold reduction in cost relative to NGS-based amplicon sequencing. To use the tool, you start ...Analyze Complex Edits   Traditional Sanger sequencing-based analysis tools are unable to detect or analyze complex...region around the guide binding site(s). The sequence base calls from the .ab1 file are also shown above each...error-prone repair typically result in mixed sequencing bases downstream of the cut. Summary ICE generates NGS-quality...

  47. Course-Based Undergraduate Research Experiences (CUREs)

    Type
    Blog Post
    Published
    Feb. 9, 2023, 2:15 p.m.
    ... Course-based Undergraduate Research Experience (CURE). Having incorporated a discovery-based lab on planarian...and research-based learning in a year-long biochemistry laboratory : Part II – research-based laboratory...thinking skills (Knutson et al., 2010a,b). Classroom-based research projects have also been proposed as a mechanism...References Bangera, G., Brownell, S. 2014. Course-based undergraduate research experiences can make scientific... E., Kowalko, J., Keene, A. et al. 2022. Course-based undergraduate research experiences (CUREs) as a ...research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory:...Implementation of a collaborative series of classroom-based undergraduate research experiences spanning chemical...

  48. Flow Cytometry Readouts: Yes, No, and Everything in Between

    Type
    Blog Post
    Published
    Aug. 20, 2024, 1:15 p.m.
    ...positions to the base expression. When compared to the control — which should be base expression — a peak...with the negative control population (Figure 1). Based on where the GFP- population ends, you can draw ...cells and might be upregulated or downregulated based on your experiment. You can see from Figure 2 that...that determining where to place your gate based on the dot plot would be complicated.  Figure 2...   In such cases, instead of trying to quantify based on percentage positive or negative in your dot plot...populations of interest with normal gates, just based on a histogram plot instead of a dot plot. However...overlaps with the baseline suggesting this sample has a comparable expression to the baseline/negative control...

  49. Illuminating Epigenetics with A FRET Based Biosensor

    Type
    Blog Post
    Published
    Nov. 19, 2015, 3:30 p.m.
    ...of cancer through different mechanisms. New FRET based tools to study epigenetics Epigenetic research uses... Alice Ting’s lab has developed a new, plasmid-based biosensor to visualise changes in histone methylation...

  50. Hot Plasmids: FRET-Based Biosensors

    Type
    Blog Post
    Published
    May 6, 2014, 1:07 p.m.
    ...of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most notably, the Twitch.... “It gives you more information than intensity-based readouts.” Griesbeck also points out that the approach...now be applied to dozens of other published FRET-based sensors. For example, his team is now applying it...Universiteit Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular...
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