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We narrowed to 612 results for: SCR

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  1. The Open Repository of CRISPR Screens: CRISPR Screen Data in One Place

    Type
    Blog Post
    Published
    Jan. 21, 2020, 2:30 p.m.
    ... and the screen results. Screenshots updated 2024. Importantly, metadata for each screen is captured...Repository of CRISPR Screens. Imagine you’ve just discovered that your favorite gene was described in a CRISPR ...CRISPR screen data that might be able to help you out. What is the Open Repository of CRISPR Screens? The...of CRISPR-based phenotypic screen datasets. Original published CRISPR screen data is curated and deposited...BioGRID Open Repository of CRISPR Screens (ORCS) homepage. Screenshot updated 2024.   Using the Open...Repository of CRISPR Screens BioGRID ORCS currently houses over 890 CRISPR screens in 629 cell lines, each...cancer" returns 40 screens performed in 26 different breast cancer cell lines. Screenshot updated 2024. ...

  2. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    Published
    Nov. 10, 2022, 2:15 p.m.
    ...right for you. Screen variations Forward and reverse screens Forward and reverse screens involve manipulating... suppressor screens These types of screens are very similar to forward and reverse screens, except that...approaches. Screens and selections The common goals The confusion in differentiating between a screen and a ...reverse screen – all known genes are assayed for their role in a particular capacity. Drug screens, on the... time screening hits that aren’t “real” or novel for their purposes… or worse yet have a screen that doesn...phenotype. These screens employ the same downstream manipulations as a forward or reverse screen (CRISPR, drug...component. Alternative screening techniques While CRISPR and drugs dominate the screening scene, there are ...

  3. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    Published
    Feb. 4, 2025, 2:15 p.m.
    ...variety of screens, including interference, activation, and knockout. Most CRISPR screens rely on lentiviral..., AAV vector screens may be AAVantageous!       In vivo, in vitro, ex vivo CRISPR screens can be carried... For in vivo screens, mice are directly infected with the CRISPR library. In vivo screens can become costly...CRISPR Delivery Genome-wide Screening Using CRISPR Performing In Vivo CRISPR Screens Using the FITS Approach...Forward genetics screens are a valuable part of the molecular biology toolbox to identify new target ...understand the intricacies of molecular pathways. These screens have gotten larger, easier, and more comprehensive...CRISPR-based technologies over the past decade. CRISPR screens are easily customizable and enable the targeting...

  4. Tips for Improving Your Next Manuscript

    Type
    Blog Post
    Published
    Nov. 22, 2016, 3:30 p.m.
    ...improve your next manuscript 1. Be consistent with your figures The figures of a manuscript are the result...introduce the reader to the manuscript and engage them in the material. So the manuscript should be able to be... When Publishing a Scientific Manuscript Writing Scientific Manuscripts: Literature Searching, Reading... a publication. There is an art to composing manuscripts and the ASM Scientific Writing and Publishing...the do’s and don’ts of that week’s piece of a manuscript. The webinars were my favorite part due to the...consisted of reading that class’s section of a manuscript and critiquing it, were not too laborious, but...helped build my understanding of how to generate a manuscript. At the end of the online course, we were given...

  5. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    Published
    Aug. 18, 2015, 2:30 p.m.
    ...genome-wide screens? The goal of a genome-wide screening experiment is to generate and screen a population...the steps involved in a CRISPR screen? Performing a forward genetic screen using CRISPR libraries is a multi-step...sorting and screened for a particular phenotype. For example, mutant cells can be used in drug screens to identify...means to carry out so-called “forward genetic screening experiments”, it is certainly the most robust....libraries are being used to perform genome-wide screens and highlight some of the reagents that have been... different genomic target. The goal of a CRISPR screen is to use Cas9 and a pool of gRNAs to identify ...CRISPR can be readily scaled up for genome-wide screening due to the broad range of potential target sequences...

  6. Plasmids 101: Blue-white Screening

    Type
    Blog Post
    Published
    June 4, 2015, 1:03 p.m.
    ...pUC19, and pBluescript are a few common vectors. Blue-white screening is just that – a screening process...insert (blue-white screening). Other types of screening methods Although blue-white screening is probably the... that! (To read about other screening strategies, check out our screening overview blog post.) *Note:...good, but how does this help us screen plasmids? Blue-white screening in the lab Scientists discovered...the medium to ensure transcription of the lac operon.   Tips for blue-white screening Use a good control...can talk about an even more specific method of screening your cloning reaction. Being able to select for...Note: Two Popular Vectors for Blue White Screening Available at Addgene Are: pUC18 and pUC19 Let’s begin ...

  7. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ...CRISPR knockout screens: Full genome vs. custom screens Full genome CRISPR knockout screens have been used...generating a massive amount of descriptive transcriptomics data. Custom CRISPR screens designed to target differentially... gRNAs in a screen, to ensure that each gRNA has a good chance to contribute to the screen. In the end...Secondary screens  If you have generated a list of candidate genes from a full genome screen, a smaller...genes you’d like to investigate in your next CRISPR screen. Luckily, the Green Listed software can help you...software tool used to design gRNAs for custom CRISPR screens targeting a (long or short) list of genes provided...fantastic tools for unbiased discovery. With these screens, the investigator generates a cell population where...

  8. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    Published
    March 30, 2017, 2:30 p.m.
    ...in a sample. Digital PCR assays for mutation screening Now that we have introduced how digital PCR works...the bi-allelic mutations typically desired when screening clones for complete functional knockout of your...interested in harnessing the power of digital PCR screening to validate your genome edits, this paper provides...Remember that although digital PCR is a great screening tool, it still requires target DNA sequencing ... the exact nature of your mutations. A great description of the plethora of available sequencing options...PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells. PLoS ONE 11, e0153901–...

  9. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    Published
    April 12, 2016, 2:30 p.m.
    ...multiplexed CRISPR systems for high-throughput screening are vastly useful for mapping the combinatorial...can be used to combinatorially modify the genome, screen for a particular phenotype, and quickly profile...expression of other genetic elements including transcription factors (Cheng et al., 2014) and microRNAs (.... Hart, Traver, et al. "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer...Hiroko, et al. "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide.... Konermann, Silvana, et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...PMC4420636. 7. Ma, Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-...

  10. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    Published
    Oct. 22, 2015, 2:30 p.m.
    ...format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation...identifying prey proteins after screening. It is also possible to screen libraries of bait and prey against...your rate of interactions when screening! Tips for two-hybrid screening Minimizing false positives: Run...cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including...flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period... regardless of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists... these discoveries to create a new system for screening protein-protein interactions. In later years, ...

  11. Dos & Don'ts When Publishing a Scientific Manuscript

    Type
    Blog Post
    Published
    May 22, 2014, 4:02 p.m.
    ...help the editor handling your manuscript decide on whether your manuscript fits the scope and the interest...proofread your manuscript and help you correct any language- and spelling mistakes. A manuscript that is well...journal. Publishing via open access means that your manuscript will be available to anyone who wishes to read...read it, without them needing a subscription to the journal. An increasing number of funding bodies insist...style and the things you need to include in your manuscript. What information do I need to include in my ...in the Materials and methods section of their manuscript to make resources easier to identify, find back...back, and share. Depending on the type of manuscript you are writing, it may be useful to consult the ...

  12. Plasmid Grant to Aid Cervical Cancer Screening in Ghana

    Type
    Blog Post
    Published
    Nov. 8, 2016, 3:30 p.m.
    ...Cervical cancer screening in Ghana Gladys is currently working to enhance cervical cancer screening in Ghana....cervical cancer screening for a limited group of Ghanaian women by including PCR screening for HR HPV alongside...as positive controls in her PCR screens. She hopes that these screens will both identify individuals who...to be at high risk by these screens can then undergo more routine screening allowing for early cervical...positive controls in PCR-based cervical cancer screens. This third grant represents the continuation of...tests are commonly used in Ghana, there is little screening for DNA markers like the presence of high risk... cancer screening...

  13. Plasmids 101: Screening Strategies Used in Plasmid Cloning

    Type
    Blog Post
    Published
    Sept. 15, 2020, 1:15 p.m.
    ... your selection, then screen Why add a screen to your cloning strategy? A screen will help you more easily...a look at a few screening methods. Blue-white screen A widely used method of screening is the blue-white...In many cases, you will need a screening strategy as well. In a screen, you aren’t killing off a portion...blue-white screening blog post for more information.   Figure 1: Result of a blue-white screen. Image...that contain your desired insert? This is where a  screen comes in. Let’s take a look at All_in_one_CRISPR...vector contains the gRNA insert or not. A blue-white screen, however, can give you this information (more on...blue-white screen, which relies on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse...

  14. Performing In Vivo CRISPR Screens Using the FITS Approach

    Type
    Blog Post
    Published
    May 2, 2024, 1:15 p.m.
    ... vivo screens in naïve T cells. More broadly, this system can be used to perform in vivo screens in other...power of in vivo screens. Figure 1: Using FITS to perform in vivo CRISPR screens to assess T cell...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...However, in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine...vivo. Over the past five years, in vivo CRISPR screens have been used to interrogate T cells, an important...complex and physiological microenvironments. These screens have primarily been performed in activated T cells...naïve T cells. The Framework for In vivo T cell Screens (FITS) approach (Milling and Markson et al., 2024...

  15. Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing

    Type
    Blog Post
    Published
    May 5, 2015, 3:54 p.m.
    ...Beginner’s Guide to Writing Scientific Manuscripts. Any scientific manuscript will require numerous other references...Scientific Manuscripts by Johnna Roose Dos & Don'ts When Publishing a Scientific Manuscript by Maaike...in ‘writing-mode’ for a grant or a scientific manuscript, the reading will likely be more intense, but...need to be on a network with an institutional subscription), archived on PubMed Central, or in preprint...down your own results; are they the same as the description the authors give? Ask yourself if the results...filled to the brim with reprints and papers with scribbled notations in the margins. If you prefer, printing...of the Beginner’s Guide to Writing Scientific Manuscripts series on Johnna's blog. Thank you to our guest...

  16. Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering

    Type
    Blog Post
    Published
    May 3, 2018, 1:48 p.m.
    ...New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...Cas13d, both groups used very general bioinformatic screens that looked for a CRISPR repeat array near a putative...Like other Cas13 enzymes, the Cas13d orthologs described in these papers can independently process their... high targeting efficiency, both of which are described below. Yan et al.: WYL-domain-containing accessory...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...

  17. Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening

    Type
    Blog Post
    Published
    Feb. 20, 2014, 4:57 p.m.
    ...Two new human lentiviral CRISPR library systems described in companion papers in Science in December were...Additionally, a concurrent Nature Biotechnology article describes the development of a mouse lentiviral CRISPR library... this technology.” GeCKO library for genomic screens Using lentivirus as the delivery vehicle, Neville...to enable both negative and positive selection screening. They and their colleagues including Feng Zhang...at a time,” Wang said. “It was very difficult to screen in a pooled fashion or to go after thousands of...expression. The Yusa lab has used their library to screen for genes involved in bacterial toxin resistance... is now possible to conduct systematic genetic screens in mammalian cells,” David Sabatini, a senior author...

  18. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    Published
    Sept. 13, 2018, 1:55 p.m.
    ...biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly...similar pitfalls exist for CRISPR screens and how best to design these screens to controls for flaws. Recently...CRISPR knockout screen to analyze the following unanswered questions about CRISPR screen design. Unanswered...CRISPR/Cas9 screen design Are non-targeting guides an appropriate control? Many CRISPR screens use non-targeting...library was validated via a growth screen and a ricin toxicity screen since essential and non-essential...The library performed well in both screens, with the growth screen having a 1% false discovery rate and...shRNA library screens only identified  60% of the essential genes. The ricin toxicity screen had a 10% false...

  19. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    Published
    June 27, 2018, 1:16 p.m.
    ...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ...!  MarathonRT: A highly processive reverse transcriptase Article contributed by Will Arnold  Listen ...RTs in this regard and is even able to reverse transcribe the extremely long, highly structured HCV genome...structured template!). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other... are dependent on long, error-free, reverse transcription of RNA such as long-read cDNA sequencing and...from A. victoria the functionalized nanobodies described here can be used to target any protein fused with...unbiased discovery of proteins involved in transcriptional regulation and chromatin structure at your ...

  20. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    Published
    Feb. 24, 2015, 7:50 p.m.
    ...gain-of-function screening when combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...the next step in high-throughput, genome-wide screening,” Konermann said. “If you want to activate all...and make sure it’s working. You can only do that screen with a system you are really confident about working...Konermann and her colleagues in Feng Zhang’s lab describe just such a system, which they call CRISPR/Cas9...SAM is an engineered protein complex for the transcriptional activation of endogenous genes and it consists...The next insight was to assemble a synthetic transcription activation complex consisting of multiple distinct...

  21. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    Published
    March 31, 2016, 2:30 p.m.
    ...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...thought to destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: ...expression levels. Transcription Termination Rho-dependent termination DNA transcription Polyadenylation...both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation...sequence-based element, is to define the end of a transcriptional unit (such as a gene) and initiate the process...

  22. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    Published
    Oct. 4, 2018, 12:44 p.m.
    ...to conduct genome-wide gain of function screens. These screens can help reveal the function of lowly expressed...large-scale functional screens Read this blog post about genome-wide screening using CRISPR Resources...allowed scientists to easily perform genome-wide screens to effectively and efficiently investigate gene... per gene to increase confidence in the genetic screen. However, there are benefits to optimized libraries...resources and are particularly useful when trying to screen a limited number of cells like primary cells or... they conducted genome-wide negative selection screens with both A375 (melanoma) and AT29 (colon cancer...metrics to assess library performance in viability screens. Based on these metrics the Brunello library was...

  23. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    Published
    April 3, 2014, 8:05 p.m.
    ...initiating the transcription of your insert into RNA. In practice, the term "promoter" describes the combination...Promoter   Primarily used for RNA     transcript     Description Expression Additional considerations ...Primarily used for Description Expression Additional considerations T7 in vitro transcription/ general expression...for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on ...controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role...

  24. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ...engineered combinations of transcriptional activators to induce gene transcription at genomic sites targeted...found here. High-throughput screening strategies Although the tools described above make CRISPRa more accessible...subgenome-scale CRISPRa screening approach in embryonic stem (ES) cells to determine which transcription factors promote...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)...-optimized viral vectors, and high-throughput screening approaches. Here, we’ll highlight these recent...CRISPRa much simpler to implement in genome-wide screens over cDNA libraries. However, using CRISPRa to ...and RNA components, and several sgRNAs must be screened to identify effective ones. Simplified in vivo...

  25. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    Published
    March 17, 2016, 2:30 p.m.
    ...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...start signal sequence (gene start) and a transcription termination/polyadenylation signal sequence (gene...genome which are required for replication and transcription (Figure 1). All viral genes are removed and ...direct minigenome replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The...minigenome is capable of being replicated and transcribed in the cell when the appropriate viral proteins...EBOV polymerase replicates the viral genome and transcribes viral genes. Four viral proteins are required...required for viral genome replication and transcription: NP, VP35, VP30, and L (reviewed in 5). The RNA genome...

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

    Type
    Blog Post
    Published
    April 7, 2015, 4:21 p.m.
    ... to modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as ...for CRISPRi are described here: http://www.addgene.org/crispr/qi/ Plasmids described in  Zalatan et ...recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has...visualize the concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated...CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes. Modifying the ...synthetic RNA scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds...domains. Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent target nucleotide sequence...

  27. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    Published
    Dec. 20, 2022, 2:15 p.m.
    ... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...other. For example, the GAL4 transcription factor cannot drive transcription from the QUAS promoter, and...and the QF transcription factor cannot drive transcription from the UAS promoter. This is important because...Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the activator protein...cannot bind the promoter sequence anymore and transcription is turned OFF. Negative Repressible: A repressor..., but cannot bind the promoter sequence and transcription is ON. Once a co-repressor protein binds the...

  28. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a ...domain, which activates transcription of downstream genes. Nihongaki et al describe the targeted activation... to activate transcription. Chavez et al. 2015 extended upon these findings by screening for additional... mutagenized and screened for speed and voltage sensitivity. This hierarchical screen led to the generation...and downstream transcription activation or repression. The TCS light sensors described in ACS Synthetic...scientists have been successful in regulating gene transcription using DNA-guided optogenetic tools; however,...recognized by scFV antibodies fused to VP64 transcriptional activation domains. This system enabled the...

  29. Plasmids 101: Inducible Promoters

    Type
    Blog Post
    Published
    Jan. 18, 2018, 2:34 p.m.
    ... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...induction of a promoter. Below, you’ll find a short description of these three types of inducible promoters, ...removal of the lac repressor (lacI protein) for transcription to be activated. In the presence of lactose ...

  30. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...an alternative method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests...independently of their position relative to a transcription start site. In STARR-seq, randomly sheared genomic...contain an enhancer, it will activate its own transcription from the minimal promoter and its strength can...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...CRISPR knockout library and used this library to screen for genes involved in West Nile Virus (WNV) induced...

  31. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    Published
    Oct. 6, 2020, 1:15 p.m.
    ... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase transcription. This is done...cells. VPR Description VPR fuses a tripartite complex with dCas9 to activate transcription. This complex...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9...

  32. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    Published
    March 30, 2023, 1:15 p.m.
    ...Transcription factors (TFs) act as the gate keepers for gene expression, turning transcription on and... regulatory network. The transcription atlas and library The transcription factor library The human... TFs is a useful finding. Transcriptional program changes from this screen successfully differentiated...-scale CRISPR Cas-9 knockout and transcriptional activation screening. Nat Protoc. 2017 (4):828-863. doi...approximately 1,800 TFs, with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive...differentiation outcomes as well as global transcriptional changes. Using this road map, and what was ...from Joung et al.)    Putting MORF to work Screens and selections In its current format, the MORF ...

  33. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64 ...readily found at Addgene. Shining light on transcriptional activation using dCas9 Initial photoactivatable...early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at ...photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression...the activator into position to activate gene transcription. The researchers tested a variety of combinations...CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in...expresses the C-terminal fragment of Cas9 while transcription of the N-terminus is light-dependent (Yu et ...

  34. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ...plasmids with the original publication where you described them. Search for your article as you would search...published. Plasmids can be linked to a peer-reviewed manuscript OR a preprint. In the case of a preprint submission...to update the publication information once the manuscript is published in a peer-reviewed journal. To start...simply enter your preprint title or a general description of the deposit. Select your desired distribution...distribute, or Addgene finds your journal publication describing the plasmids, or (for deposits submitted after...selected your publication or entered your deposit description, the next page will allow you to add plasmids... article. Note that if you entered a deposit description, this will become your article title.   ...

  35. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    Published
    July 6, 2021, 1:28 p.m.
    ...themselves as RNA transcripts and then rely on a reverse transcriptase enzyme to reverse transcribe back into ...they copy themselves as RNA transcripts and then use their reverse transcriptase (RT) enzyme to convert back...mutagenesis screens that detect loss-of-function or gain-of-function mutations. In these screens, the transposons...transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters and enhancers...protein is either a transposase or a reverse transcriptase, depending on the mechanism of transposition...pol. The pol polyprotein encodes the reverse transcriptase and integrase enzymes which are required for...poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.    Figure 1: Overview of...

  36. Bioinformatics at Addgene

    Type
    Blog Post
    Published
    July 14, 2022, 1:15 p.m.
    ...copying and pasting pieces from our existing script into the script block of the Nextflow process.  Next, we...of data. In this two-part blog series, we will describe the importance of bioinformatics at Addgene and...possible solutions for each in the form of one-off scripts or programs, our goal was to find a suitable software...sequential computational tasks for which existing script(s) have been developed for a singular purpose (...sequence assembly), but the order and usage of the scripts needs to be customized in the pipeline for the ...In Nextflow, a process defines which command or script will run and can be thought of as a particular ...Prior to using Nextflow, we routinely wrote Python scripts, queried our database with SQL or Django, and executed...

  37. CRISPR Kinome Libraries Available: Pooled and Individual Plasmid Formats

    Type
    Blog Post
    Published
    Sept. 9, 2016, 2:43 p.m.
    ...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using ...at the Broad Institute, John Doench. A genetic screening project can be a tremendous undertaking, producing...producing a wall of results that can only be described as bigly. But such a project should not be undertaken...technology, gRNAs to knockout genes – are essential screening components, the model system and the assay are... in two sets, each with 4 gRNAs per kinase, so screens can be performed with either 4 or 8 gRNAs per gene...found in genome-wide libraries, and thus can be screened in pooled format at a more-typical scale of tissue.... The individual plasmids will be useful when screening for phenotypes, such as cell-non-autonomous effects...

  38. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    Published
    Nov. 3, 2022, 12:15 p.m.
    ...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...need to screen hundreds of clones looking for your edit of interest. For all of the screening strategies...phase of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base...needle in a haystack before going on to generate and screen individual clones. If a clonal line isn’t your ... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ... the minimum number of clones you would have to screen to identify a knockout clone. For example, if your...interest. Thus, to find a null clone, you should screen, at the very least, 4 clones. Did you use a dual...

  39. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    Published
    Nov. 14, 2024, 2:15 p.m.
    ...signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure... coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out ...ncRNA locus (Ho et al. 2012). For large-scale screening, sgRNA libraries are useful for targeting thousands...lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting...the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters...the removal of exons. Disrupting promoter or transcription start site function would also help to reveal...

  40. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening - An excellent protocol paper by...Feng Zhang lab describing construction, lentiviral production, transduction, screening, and validation...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017 Apr;12(4):828-...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...the library sample, and any other information or scripts provided by the depositing lab for data analysis...

  41. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    Published
    Dec. 10, 2019, 1:58 p.m.
    ...activation of transcription in bacteria by co-opting σ54-based transcription. Transcription from σ54 promoters... as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening of cells...pooled libraries are popular tools for genetic screens, enabling genome-wide interrogation of the genes...required for cellular processes. For a successful screen, it is necessary to identify the gRNAs that elicit...been created in lentiviral systems for mammalian screens, which enables researchers to maximize the chance...are poorly transduced by lentivirus, and thus screening with available CRISPR libraries was not possible... from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation...

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

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...silence the target gene(s) either transcriptionally or post-transcriptionally. In plants, different dicer-like...ones? Here, I’ll cover some screening methods to identify them. Visual screening Regardless of the RNAi-trigger...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ...alternative start codons, and/or the phenomenon of transcriptional adaptation (Smits et al., 2019; Sztal and Stainier...RNAi in the context of a high throughput survival screen found that either technology was equivalently precise... researcher, the traditional hpRNA and visual screening method was limited in two substantial ways: variable...

  43. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ...expanded the genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability ...genetic screens, and this optimized toolkit furthers Cas12a’s potential in future genetic screens.   DeWeirdt...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...Cas12a has generally been underutilized in genetic screens because of its lowered editing rate. This research...aimed to increase the potential of Cas12a genetic screens by optimizing enzyme activity using an enhanced...

  44. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    Published
    April 23, 2018, 2 p.m.
    ...estradiol controlled transcription factor and a ZIF268/progesterone controlled transcription factor as well ... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...Tyler J. Ford Listen to the Synthetic Yeast Transcription Factors Podcast Segment Researchers from the...deposited plasmids encoding synthetic yeast transcription factors that can independently control expression...expression of your genes of interest. These transcription factors consist of the GAL4 or ZIF268 DNA binding...domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate ligand...addition to plasmids encoding these synthetic transcription factors, the El-Samad lab has deposited plasmids...

  45. All in a Twist: dsRNA

    Type
    Blog Post
    Published
    Feb. 2, 2023, 2:15 p.m.
    ...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...will dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly...thought to be associated with bidirectional transcription. In an uninfected cell, dsRNA rarely circulates...short, dsRNA molecules designed to degrade mRNA transcripts. RNAi activates the RNA-induced silencing complexes...elements can generate dsRNA by bidirectional transcription or as inverted repeats. These elements are almost... produce dsRNA as a result of bidirectional transcription of their circularized DNA. These dsRNAs are ...structure produced is known as an R-loop. During transcription, RNA is synthesized from a DNA template, inherently...

  46. Plasmids 101: Modular Cloning

    Type
    Blog Post
    Published
    March 12, 2024, 1:15 p.m.
    ...into… Transcription units (TUs), which are functional units composed of multiple parts transcribed together...a protein that regulated its own transcription, or the transcription of another gene? Now you have a genetic...specifically refers to the organizational schema described above, and not any particular cloning technique...hierarchical levels: individual DNA parts, transcriptional units, and multigene constructs. Let’s start...start by examining individual parts and transcriptional units. Individual parts are defined DNA sequences...assembled into larger functional units which are transcribed together. Image courtesy of Robert Hurt. ... with DNA, and these complexes regulate the transcription of specific genes. What if one of those genes...

  47. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being...being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific...potential options and cloning methods, but please scroll down for plasmids suitable for other model systems...system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter... Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric nuclease...like AAV. In addition to the mammalian option described below, plasmids for making polycistronic gRNAs...

  48. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers...TALEN-based LITE system, which uses light to regulate transcription, also includes light-regulated histone methyltransferases...check out our CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused...enhancer regions. These marks are associated with transcriptional activation. Created with BioRender.com. ...Tet1-CD to decrease methylation and activate transcription. A lentiviral vector with the same modifier,... demethylation is associated with increased transcription. Created with BioRender.com. Tet1 initiates...

  49. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    Published
    Aug. 18, 2016, 2:30 p.m.
    ...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use...the best uses for Cas9 activators is in genetic screening. gRNAs targeting every gene in the human genome...interactions. Additionally, large scale CRISPR-based screens can be used to gain insight into protein structure...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides by...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568:511–516 . https://doi.org/10.1038/s41586...

  50. Protein Tagging with CRISPR/Cas9: A Conversation with Mendenhall and Myers

    Type
    Blog Post
    Published
    July 28, 2015, 2:30 p.m.
    ... pair has focused considerable attention on transcription factors, starting with an established method... relies on antibodies to recognize and bind transcription factors directly, but that’s a slow-going process...ENCODE project is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the... ChIP-seq, but it’s really hard for lots of transcription factors. Our groups got together to adapt CRISPR...big difference. We can now look at many more transcription factors. Addgene: How does the system work? ...second plasmid to target an epitope tag onto the transcription factors. By the way, you can actually use this... in all kinds of different assays, not just transcription factors. The second vector is a homology directed...
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