Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of the Addgene plasmid sequence database.

Information

In Category

We narrowed to 1,100 results for: Ide

Showing: 241 - 280 of 1100 results

  1. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...immunohistochemistry, and are summarized in the table below:   ID Cv (Capsid) FLAG Tag Terminus 79629 (N) and 79628... Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-...DHS-seq, FAIRE-seq, and CHIP-seq), genome-wide identification and quantification of enhancer activity have...RNA-guided targeting mechanism. However, the laborious construction of repair templates and guide RNA ...: Targets of RNA-binding proteins identified by editing Identifying targets of RNA-binding proteins (RBPs...region sequencing) that tests enhancer sequences outside of their endogenous chromatin environment and relies...screen the entire genome for genes that regulate a wide variety of phenotypes, including but not limited...

  2. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169...Cas9, been identified. In Rauch, et al. 2016 the Joseph Bondy-Denomy lab at UCSF identifes four proteins...our hot plasmids articles. These brief articles provide a synopsis of a plasmid or group of plasmids' functions...may be preferred for their intended uses, they provide a well-defined framework for the production of ...available at Addgene, and the Doyon lab has also provided a detailed protocol at Protocol Exchange. Plasmid...editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated with... created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently tested...

  3. Video Library

    Type
    Protocol
    ...questions. Protocols Video protocol guides for standard laboratory procedures. How-To Videos Screencapture walk-throughs...Protocol How-To Videos (Link opens in a new window) Video Link Description Related Page MTA Guide - Requests... Educational Resources Addgene Videos Addgene Videos Browse Addgene's collection...collection of video content. Find video protocols, tips on using Addgene's resources, and career advice. For...walk-throughs to help guide you through Addgene's online resources. Career Videos Get career insight and...to Deposit Your guide to depositing plasmids with Addgene Depositing Page Career Videos (Link opens in ...A collection of Addgene's video content, including how-to screen captures, lab procedure protocols, and...

  4. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

    Type
    Blog Post
    ...(See section below.) For guide design, we decided to adapt the popular guide design tool Crispor by Max...which the guide binds. However, if the PAM resides in a coding region, the mutation may cause side-effects...optimal mutation in CrispyCrunch: If the guide PAM resides outside a coding region in a UTR, CrispyCrunch... experiments designed outside CrispyCrunch, or you can input pre-existing guides for primer design. The...return guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide design... summary per batch. Ranking guides for HDR in-depth In other tools, guides are ranked by specificity of...specificity of a guide to the target. The CFD score ranges between 0 and 100 for each guide, with 100 being...

  5. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    ...genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by sequencing (GUIDE-seq) ... Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing Provided 20% ...PMC3782611. 8. Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by ...gel electrophoresis. Surveyor™ and T7E1 have been widely adopted due to their relative simplicity and low... problem with these assays is that they do not provide sequence-level data. They also have a limit of ...sequencing. Used in concert, these approaches can provide the researcher with a reasonable level of certainly...off-targets. This is a valuable step toward enhancing confidence and reproducibility of a study’s findings. Table...

  6. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...Views Guides CRISPR Guide Lentiviral Guide AAV Guide Optogenetics Guide γ-Retrovirus Guide Protocols... popular videos are protocol videos, where showing exactly how to execute a step can provide needed clarity...accessible, due to a wide range of logistical barriers. Addgene worked to identify, reduce, and remove ...allowing people to access a wide catalog of plasmids from all over the world. This idea, that available and accessible...Addgene distributes to per year, 2005-2023. We identified and offered a number of solutions that increased...over half of our orders coming from researchers outside the USA, having some materials available to order...consistently accessible, wasn’t an option. So we identified countries that would benefit from a third-party...

  7. A Look at Addgene's QC Process

    Type
    Blog Post
    ...plasmids we distribute to scientists worldwide. Depositing scientists provide different types of data for each...compared to the information provided by the depositor. If a depositor provides a sequence for the plasmid...sequences without a reference; and identifying mixed plasmid and nucleotide populations. All of these topics...if one was provided. If no reference is given, de novo assembly in Geneious can still provide additional... their presence with confidence. If this approach does not resolve the identified differences, the plasmids... both human and biological. Even the most high-fidelity polymerases can make mistakes and lead to the ...plasmids in our collection. This standardization provides security for requestors, ensuring that the plasmid...

  8. Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

    Type
    Blog Post
    ...DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the RNA guides employed...remains faithful to its guide DNA and cannot switch guides. While this fidelity would prevent other small...24 base DNA guides may be more user-friendly, since they can be ordered as oligonucleotides and should ...gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA guides must ... tested if NgAgo preferred certain guide sequences - using 5 guides/gene for 10 genes, they did not observe... the DNA guide impaired NgAgo activity, with three mismatches abolishing activity. One-guide faithful...important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also...

  9. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...similar to TIDE known as TIDER (Tracking of insertions, deletions, and recombination events). TIDER can screen...both trace files to the TIDE tool online along with your sgRNA sequence. The TIDE software will generate...off-target effects with Sanger sequencing, provided you have some idea of where in the genome you might expect...knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et al., NAR). This technique uses the... deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files of WT and edited...input population or clone.   How to perform a TIDE First, use PCR to amplify the target region of interest...graph representing all insertions and deletions identified within a given genomic window, along with the...

  10. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...with peptide/protein analyses using ELISA. Peptides 72:4–15 . https://doi.org/10.1016/j.peptides.2015.04.012...vary the acrylamide content of the gel. For small proteins, use a higher percentage of acrylamide to increase...usually composed of a nitrocellulose or polyvinylidene difluoride (PVDF) in a step called protein transfer...they provide qualitative measurements. If you require precise quantification of a protein, consider using...immunoprecipitation and pull-down assays.  Westerns are divided into two categories, native and denaturing. In ... protein mix by size Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first...agent, typically containing thiols, to cleave disulfide bonds. Load the samples, one sample per lane, ...

  11. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ...Having answer slides ready after your formal conclusion slide will also help with your confidence. It is best...suspense. Notice the simplicity of their slides and how the slides complement the spoken message. Learn from...speak to start creating slides or thinking about content. Create a Google.slides or Powerpoint file right...this might sound condescending, consider explaining the term on a slide but not reading the explanation...everything I allow 1 slide for every 1 minute of talk length, and then I cut a few more slides. Do not run over...always be a future boss in the room, so it is a good idea to start thinking about public speaking early and...talks for non-academic settings. Many of these guidelines will apply to any type of science presentation...

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

    Type
    Blog Post
    ...overhangs with the same three nucleotides in a row; (4) no more than two nucleotides in the same position; (...that high-fidelity reactions could be done using overhang sets that broke rules 3—5. Fidelity, it turns... design high-fidelity Golden Gate Assemblies and overhang sets: NEBridge Ligase Fidelity Viewer™; NEBridge...reactions. NEBridge Ligase Fidelity Viewer This tool allows a user to check fidelity of an overhang set by ...remarkably high 71% fidelity, while the 52-piece assembly of a lac operon dropped to 49% fidelity, with significant...be strictly followed in order to generate high-fidelity reactions. (Need a refresher? The five rules are... a single tube. Their assembly had a predicted fidelity of 71% (Pryor et al., 2020). And in 2022, using...

  13. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    ... requires that you provide two out of six forms of documentary evidence: Evidence of receipt of major...obtain permanent residence. The recently established case Matter of Dhanasar provides the three-prong ...field Evidence of original scientific or scholarly research contributions in the field Evidence of authorship... to convey general information only and not to provide legal advice or opinions. The contents of this ...advice on specific legal issues.  The permanent residence “green card” process is a necessary and often ...uncertainty associated with filing a permanent residence case with United States Citizenship and Immigration...national researchers who require visas, permanent residence or citizenship to continue their work in the US...

  14. Of Myc and Men

    Type
    Blog Post
    ...defined peptides. Munro and Pelham’s first published peptide tagging experiment involved a peptide from ...with unrelated peptides that already had high quality antibodies available - aka peptide tagging.  Key ...bound to the mid-protein peptide, while three bound to the C-terminal peptide. One of these C-terminal... of these new antibodies and identified Myc1-9E10 and its target peptide as a suitable pair for protein...from the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino...could be quite small. However, substance P has an amide group added to its C-terminus post-translationally...critical for antibody recognition. As such, the peptide could only be added to the C-terminus of proteins...

  15. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking. The...One 2013 Oct 7;8(10):e76117. Irreversible peptide-peptide ligation using SpyLigase Building off their...that promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against...scientists to use CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries... to browse CRISPR plasmids, watch informational videos, download protocols, and more! Looking for background... CRISPR technology? See our improved CRISPR-Cas Guide. Wang et al., Science. 2014. Jan 3;343(6166):80...

  16. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ...libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined ...10-6 mutations per nucleotide per generation vs. 10 x 10-10 mutations per nucleotide per generation of ...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...O., et al. "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window... changes to a DNA sequence can help scientists identify and/or facilitate the evolution of new phenotypes...method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...

  17. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    ...even single mismatches within a CRISPR guide can prevent the guide from binding to the genome or prevent...thetargeting sequences of guides across 8 CRISPR libraries. Samples are divided into those of African ancestry...approximately 20% more single nucleotide polymorphisms (SNPs) that map to guides in the Broad’s Genomic Perturbation...accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’ genomes... CRISPR screens in DepMap to see if they could identify the ancestry of the cell lines (European, East...germline genetic variations. They then wanted to identify cancer-associated genetic dependencies associated...relationship between the genetic dependencies they identified and their associated ancestries. While there ...

  18. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...adapted "self-cleaving" 2A peptides into their muliticistronic vectors. These peptides, first discovered in ... these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at...cleavage with some of these peptides, no consensus has been reached on which peptide works best. Likely the...from a single transcript. The two strategies most widely incorporated into plasmids for research purposes...the specific genes cloned into the vector.3  2A Peptides To overcome some of the disadvantages of the IRES...between the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the ...Proline.  The table below lists the four common 2A peptides employed by scientists. 2A cleavage is universal...

  19. Tips for Using BLAST to Verify Plasmids

    Type
    Blog Post
    ...menu. Typically, we use the default nucleotide database “Nucleotide collection (nr/nt)” as it contains ... the Basic Local Alignment Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing...reference sequences, such as full plasmid sequences provided by the laboratories that deposit their plasmids...programs available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp...BLAST (blastx). NCBI has a terrific getting-started guide for BLAST, which includes a simple explanation of...BLAST search pages. At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as...ORFs) and determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs...

  20. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...A,T or C – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations...mutation), blocking the guide RNA binding site works well. However, since some guide RNAs may tolerate a ...end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred...repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the intended sequence...efficiency.   But what’s better: PAM-blocking or guide RNA blocking mutations? This depends entirely on...however we have also demonstrated the efficiency of guide RNA blocking mutations. So if the locus does not...while taking advantage of the improved efficiency provided by blocking mutations. Essentially, you:  Step...

  21. How Addgene Creates Content

    Type
    Blog Post
    ...social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to provide informative...using the example of our Antibody Guide, which was published alongside our new antibody services in the... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise.... Since our guides are heavily used, our graphic designer made most of the Antibody Guide figures. This...topics for the Addgene blog? Or why we make certain videos? In this post, we invite you behind the scenes ...you, whether you realize it or not! Many of our ideas come from social media conversations, conferences... all of our frequently asked questions!    Identify needed content Let’s take a look at how we create...

  22. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    ... particular, one slide of the presentation made a lasting impression on me. The slide showed a complex...approach is to identify an appropriate list of genes to target. Thus, it is always a good idea to keep a bioinformatician...Sweden. It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you...screens targeting a (long or short) list of genes provided by the user. This approach is useful for several...effective by world class CRISPR labs.  Check Out This Video to See How Green Listed Works at a Glance!  CRISPR...subsequently sequenced by next generation sequencing to identify which gRNAs are differentially expressed in the...to dissect a complex signaling pathway, and to identify what part of a pathway to inhibit to block the...

  23. Building Global Connections with the International Mentorship Program USA-EUROPE

    Type
    Blog Post
    ... more fully identify with and be inspired by the mentor’s successful example. For evidence of this, you...Valencia): “IMP showed me a wide range of professional opportunities that are outside of our typical boundaries... Program (IMP) USA-Europe was created. IMP as an idea was conceived and developed by its Director, Dr.... closer than ever (although not physically) the ideas and experience from talented professionals to their...together with the universities, a bright future. IMP provides matched one-on-one international mentorship, with...globalized world where individuals can exchange ideas, network, collaborate, and innovate internationally...offered by programs like IMP USA-Europe not only provide the specific technical and logistic support to ...

  24. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    ... that you provided for each sample. Guide Target - This is the user-defined 17-23 nucleotide sequence ...sequencing files and provide basic information such as your sample names and guide sequences, and ICE will...which also includes our CRISPR knockout guide design tool, guide validation tool, and guaranteed editing... Lead at Synthego. Article source: Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE on...editing field by making experimental workflows considerably easier, faster, and more efficient than previous...examined ICE analysis of Sanger sequencing data alongside the analysis of NGS-based amplicon sequencing ...time or as a batch of hundreds, and indicate the guide RNA sequence you used. The ICE tool will calculate...

  25. Story of a SynBio Startup: RevBio's Epiphany (or Lack Thereof)

    Type
    Blog Post
    ...joint. Silly ideas (mining local salt deposits to sell at farmers markets), and serious ideas (how to stop...for the future. From good ideas to a SynBio startup At some point, our ideas got pretty good. We started...journal clubs outside of work. Our scientific training was put to use as we explored ideas, planned out...we could do to make ourselves marketable to the wider world and occasionally thinking about how we could...establishment hiring system altogether. We had a lot of ideas about things we could do for work, and they weren... together we make a pretty good team. We have confidence that we can take on any challenge and quickly...marketable product. During this process, we had an idea that survived the literature review, survived technical...

  26. Evolution of Lab Techniques

    Type
    Blog Post
    ...chain-terminating or dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string...Sanger sequencing uses modified nucleotides, called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl...added and when. Compiling the identity and location of a given nucleotide, a researcher (or a simple computer...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...Edward M. Southern (1), can be used to detect DNA identity, size, and abundance. If a scientist wants to ...gene mutation, southern blotting can be used to identify the presence of that mutated gene. In southern...One example of southern blotting being used to identify a gene mutation is the genetic test used for sickle...

  27. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    ...whenever applicable?Against this, consider the cost of an accidental release. Science relies on popular... DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate flanking homology...homology into a germline cell can create an RNA-guided gene drive. In principle, this event could happen even...available. Consequently, the best way to avoid accidentally creating a gene drive is also the easiest: don't...barrier protocols, they can reduce the risk of accidental escape to a negligible level. One major reason...publish the concept and likely capabilities of RNA-guided gene drives in the summer of 2014 (after extensive...hope was that anyone else who came up with the same idea or wanted to build a gene drive in a model organism...

  28. Components of CRISPR/Cas9

    Type
    Blog Post
    ...to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA provided that target...the components necessary to: 1. Bind to a guide RNA The guide RNA, which will be described in more detail...of the site targeted by the guide RNA. In the absence of either the guide RNA or a PAM sequence, Cas9 ...synthetic guide RNA or gRNA (sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its...into one RNA sequence known as the guide RNA (gRNA) or single guide RNA (sgRNA). Throughout the remainder...Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After ...Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9...

  29. Bricking Science: Portraying Scientific Reality Through LEGO

    Type
    Blog Post
    ...Producing stop motion videos is very time consuming; producing a 1-2 minute video takes an entire week ...with the images thereby making the videos more real. To give you an idea of the amount of work, see how many...’s gotten easier now that I identify with the main character in the video. We’ve grown attached to the...Dalila Cunha de Oliveira. Bricking Science is an idea built, literally, 'brick-by-brick' to introduce ...mysterious source of failure the lab gnome. See this video where he/she/it/something is captured on camera:...most people don’t get to experience this very human side to scientific research. Find Protocols Lab Tips ...was born. We chose the stop motion format for our videos as it is the most approachable for LEGOs. Stop ...

  30. BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development

    Type
    Blog Post
    ... development. Paula Videira: Congenital disorders of glycosylation Paula Videira is an Assistant Professor...been working with the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases...through the BeHEARD Award. Congratulations to Paula Videira, Michael McMurray, and Richard Gronostajski who...steps in the glycan biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the...) activity due to mutations in the PMM2 gene. Videira’s group recently reviewed and reported that PMM2...studied and understood.  In line with this, Prof. Videira and collaborators will use Addgene plasmids to ...altered in PMM2-CDG. These studies are expected to identify new therapeutic targets/solutions and enable scientists...

  31. Antibodies 101: Validation

    Type
    Blog Post
    ...the bulk of the peptides identified to be from the target protein. This approach is ideal for validating...the sample was identified as your target protein, allowing you to proceed with confidence.  Share your ...experiments that you’ve been planning, it is a good idea to pause and make sure that your antibody has been... appropriately validated so that you can have confidence in your results. But what is appropriately validated... This post will go over some of the general considerations you should review before using an antibody.... that your antibody is not binding to proteins outside of your target protein. Show suitability for an...There are a number of resources available that provide in-depth discussions on strategies for antibody...

  32. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...If you have a wide variety of protein amounts on the same blot, you may want to consider using a different...online/ask an expert Review protocol videos for a visual guide to good technique High background ...lightly marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and... buffers and strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing...re seeing protein aggregation, you may want to consider changing your incubation temperature. Some proteins...aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, ...If you need very accurate protein measurements, consider using an unstained protein ladder, which will ...

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

    Type
    Blog Post
    ...Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 orthologue... essential genes in a wide variety of bacteria including in pooled, genome-wide, formats. Interested in...dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that further improves... Then, pCRISPR is introduced along with an oligonucleotide repair template. Through recombineering, the...in Streptomyces  Streptomyces bacteria produce a wide variety of bioactive natural products. To easily...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) and the Zhang lab's system...select genes in E. coli. Furthermore, to demonstrate wider applicability they demonstrated success of Cas9 ...

  34. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    ...our CRISPR guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease...crRNA/tracrRNA hybrid guides needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than...Cpf1 functions differently. Since Cpf1 loci are widely distributed across bacterial species, Zetsche et... crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With...SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these...and off-target efficiency When Cpf1 was first identified, we didn't know much about its on-target and ...

  35. With an Eye Towards the Future, We Look Back at the March for Science

    Type
    Blog Post
    ...a march in support of science, I loved the idea and decided to go. At my local March for Science in Albany...advocate for scientist participation in government, evidence to inform policies, a protected place to work,...hired hinges on performing near miracles. When evidence does not affirm our perception of how things are...scientists are tempted to seek out alternative evidence and discard inconvenient findings. Yet, we cannot...scientists to rally in Ottawa to protest the “death of evidence,” and to speak out in unprecedented force during... and actions beyond marches, as well as allies outside the scientific community. Vanessa Sung is currently...Centre at McGill University in Montreal, Canada.  Outside the lab, she dabbles in science policy, public ...

  36. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This...discuss differences between these approaches, and provide updates on how best to design gRNAs. You can also...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should...since potential target sites occur ~1 in every 8 nucleotides, restricting gRNAs to 5 – 65% of the protein ...with a PAM preference of NGG, is still the most widely-used Cas enzyme, the development of SaCas9, NmeCas9...

  37. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...usually pick 3–6 guides around the region to find the most efficient guide (most guides would work, but...and the concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence...HR template vector design guidelines? A7: A few notes below are considerations for designing HR donor. ...plasmid, clone in your target guides, test run them, and then select the best guide to co-transfect in HR donor...100 bp away, ideally less than 10 bp away. For small (<50 bp) changes, you should consider using ssDNA...for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA... drive expression, is it necessary to add a G nucleotide to the start of my target sequence? A3: The human...

  38. Build Your CRISPR Vocabulary

    Type
    Blog Post
    ...programmable guide sequence as the spacer and the remaining sequence as the scaffold. gRNA: "Guide RNA" is ...between the guide and target in order to cut DNA, whereas some mismatches can be tolerated outside this region...CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences...includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs both functions. Cas9 cuts both...all have in common is the ability to use an RNA guide to target other nucleic acids—usually DNA—to cut... sequences in the CRISPR array that will become guides for future targeted degradation. Before integration...foreign DNA that Cas proteins recognize using an RNA guide. Non-target strand: The paired strand of foreign...

  39. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ...do you identify the actively silenced ones? Here, I’ll cover some screening methods to identify them. ...RNAi-trigger type you decide, any RNAi experiment benefits from an easy method to identify and quantify actively... example, while gene knockouts have long been considered the “gold-standard” for LOF studies, the high...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation...the importance of using orthogonal methods to confidently conclude the function of a gene of interest. ...RNAi activation. A typical RNAi experiment can be divided into three fundamental steps: creation of the ...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based...

  40. Hot Plasmids February 2024

    Type
    Blog Post
    ...with Peptide-Assisted Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and...gene delivery capsids previously identified in rodents have not been widely successful in primates. The authors...articles.  Here's what you'll find in this post: Peptide-assisted genome editing for primary cells Monomeric...delivery Recombinant anti-FLAG tag antibodies Peptide-assisted genome editing for primary cells By Susanna...and co-incubate it with an “assist peptide” that facilitates endosomal escape. Combining Cas9-PAGE with...with a 30-minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused...reporter (mChe), with co-incubation with assist peptide (AP). C) Workflow for Cas-RNP-PAGE editing of primary...
Showing: 241 - 280 of 1100 results