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We narrowed to 94 results for: sgrna

Showing: 1 - 20 of 94 results

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

    Type
    Blog Post
    ...CRISPRko sgRNA library The Brunello library consists of 77,441 sgRNAs with an average of 4 sgRNAs per gene...contains two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able...containing only 3 sgRNAs (setA) also outperformed CRISPRi libraries with 10 sgRNAs in the sgRNA metrics, confirming...2014 to predict sgRNA activity. In this study scientists examined approximately 1,900 sgRNAs and identified...subsampling analysis of the sgRNAs, Brunello even with only a single sgRNA outperformed another CRISPR...CRISPR library with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries. Thus, the Brunello... CRISPRko sgRNA library as pooled library or lentiviral prep! Dolcetto: human CRISPRi sgRNA library While...

  2. Visualizing Genomic Loci with CRISPR-Sirius

    Type
    Blog Post
    ... labeling. (A) Diagram of sgRNA-Sirius-8XMS2, sgRNA-Sirius-8XPP7, and sgRNA-Sirius-4X(MS2-PP7) showing...and Pederson created another sgRNA containing the PP7 hairpin sequence (sgRNA-Sirius-8XPP7) to recruit GFP...loci of interest. To determine how the sgRNA-Sirius-8XPP7 and sgRNA-Sirius-8XMS2 scaffolds performed, they...detection With a third sgRNA containing alternating MS2 and PP7 sequences (sgRNA-Sirius-4X(MS2-PP7)) that...Two MS2, PP7, or boxB sequences inserted into the sgRNA generates blue, green, or red colors for labeling...combination of MS2, PP7, or boxB sequences in the same sgRNA will generate additional colors. Image from Ma et...CRISPR-Sirius into cells. They named this guide sgRNA-Sirius-8XMS2 which will recruit the HaloTag with...

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

    Type
    Blog Post
    ...new sgRNA for your experiment, select from a variety of empty sgRNA vectors and design your sgRNA targeting...other.  If having two sgRNAs isn’t feasible for your experiment, ensure your sgRNA sequence has little ...next? Designing or selecting a sgRNA Addgene may carry a validated sgRNA for your target gene.  Validated...serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets two conditions:  1) The sequence...off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking” Cas9, in which...In order to produce a double stranded break, two sgRNAs must be used to target two Cas9-nickases (Cas9n...SpCas9. The drawback of this approach is that two sgRNAs targeting opposite strands must be available in...

  4. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    ...of what determines good sgRNA design. Incidentally, the first software for sgRNA design (by Hsu et al.,...technical expertise, thoughtful single guide RNA (sgRNA) design, and the abnormally low GC content of the...without computational support. Indeed, rational sgRNA design is not possible without relying on some kind...end of 2014, I began developing software to make sgRNA design accessible to all. At the time I thought ...  Breaking down the barriers: My view on current sgRNA design tools The goal of this post is to provide...software can help. The foremost handles the design of sgRNA and represents the lion’s share of the currently...however, not a focus of this blog post. I separate sgRNA design tools into database and de novo solutions...

  5. Build Your CRISPR Vocabulary

    Type
    Blog Post
    ...require a tracrRNA. sgRNA: “Single-guide RNAs” were invented by synthetic biologists. An sgRNA combines a crRNA... tracrRNA pair or an sgRNA. It is often used interchangeably with the term sgRNA. Seed sequence: The 8...complementary DNA sequences. In laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “... on the 3’ end of an RNA guide (either crRNA or sgRNA). Cas9 requires perfect complementarity in this ...

  6. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...transcription at genomic sites targeted by specific sgRNAs. Its RNA-guided nature makes CRISPRa much simpler...all the protein and RNA components, and several sgRNAs must be screened to identify effective ones. Simplified...constrained by AAV packaging limits (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression...expression of the SPH activator paired with AAV sgRNA delivery make the SPH mouse an attractive tool to...Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized viral vectors...lentivirus system pairs ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression...lentivirus system with ubiquitous expression of sgRNA (top) and neuron-specific expression of dCas9-VPR...

  7. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    ...thousands of base pairs. Cloning sgRNAs Generating constructs to produce sgRNA for CRISPR experiments can be... plating, screening and generation of sgRNA. Generation of sgRNA libraries, with maximum complexity, is...constructs with multiple point mutations Cloning sgRNAs Production of Yeast Expression Cassettes Generating...synthetic oligo to generate a single-guide RNA (sgRNA). Using only one single-stranded oligo, instead ...to create a double-stranded oligo. Generating an sgRNA construct becomes as simple as combining your favorite...

  8. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...Cas9 expression cassette and sgRNA expression cassette. The target sgRNA can be cloned directionally into...you are happy with your selection, ordering the sgRNA is simple and easy. Several companies supply gRNA...reconstitution. When the target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair...cassette. (b) pCAG-EGxxFP target plasmid and pX330-sgRNA plasmid (Mashiko et al. 2013). The pCAG-EGxxFP target...approximately 500 bp genomic fragment containing the sgRNA target sequence can be placed in multi-cloning site...was co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T cells. The fluorescence intensity.... Using this technique, you can assess several sgRNA’s at the same time, and choose the most effective...

  9. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    ...molecular cloning techniques to determine CRISPR/sgRNA efficiency of an experiment and confirm the correct...enables scientists to both quantify Cas9 activity and sgRNA efficiency as well as enrich for mutants through...2014). Thus it is essential to identify the best sgRNA to ensure success in your CRISPR experiments. But... the N-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired...expression using FACs sorting, confirming yet again that sgRNA structure and genomic recognition sites play a large...to use GEmCherry2 to identify the most efficient sgRNAs to target a specific locus to create a knock-out...

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

    Type
    Blog Post
    ... activator (CRISPRa) sgRNA library uses the sunCas9 system and contains 10 sgRNAs for each transcription...nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop...gain-of-function screening when combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited a high...transcriptional repressor (CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977...further optimize those now. They are also developing sgRNAs that will enable even more fine-tuning of gene ...

  11. Design Tips for Prime Editing

    Type
    Blog Post
    ...your tool of choice. Nicking sgRNAs PE3 and PE5: These tools use an sgRNA to guide nicking of the unedited...prime editors bound to sgRNAs only drive nicking (right). Start with nicking sgRNAs ~50 bp from the prime...bases in this location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. ... PE3b and PE5b: In this approach, the nicking sgRNA is designed to bind only after the edit is installed...the PE3/PE5 approach. Figure 7: Nicking sgRNAs can be designed to target edited DNA, so that nicking...

  12. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ...inducible lambda-red gene, and an IPTG-inducible sgRNA sequence targeting the pMB1 replicon on pTargetF...addition to the pMB1 replicon, pTargetF contains the sgRNA targeting the genomic site of interest. The donor... To use this system, you’d first introduce your sgRNA sequence into the pTargetF construct and then introduce...cured from the cell by inducing expression of the sgRNA that targets the pMB1 replicon, resulting in cutting...subsequent introductions of pTargetF containing another sgRNA targeting another site of interest can be used to...editing process begins with the addition of the sgRNA sequence in pEcgRNA. Next both plasmids are transformed...counterselection marker and will be replaced by the sgRNA targeting the genomic site of interest. We compared...

  13. Zhang Lab CRISPR Page

    Type
    Collection
    ...described here. #60224 - AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR...lentiCRISPR - sgRNA and SpCas9 together 2 vector system - lentiCas9-Blast and lentiGuide-Puro (sgRNA) Browse...CMV-driven SaCas9; U6-driven sgRNA 61593 : PX602; TBG-driven SaCas9; U6-driven sgRNA 61592 : PX600; CMV-driven... SaCas9 sgRNA can be co-transfected as PCR amplicons using a protocol similar to SpCas9 sgRNA PCR (Ran... pAAV-U6::sgRNA(SapI)_hSyn-GFP-KASH-bGH (SpGuide acceptor). This is a AAV plasmid for sgRNA cloning. GFP-KASH...implemented by delivering Cre and sgRNA to a Cre-dependent mouse or sgRNA to a constitutively Cas9-expressing...control for AAV-KPL; sgRNA targeted to LacZ, plus luciferase-2A-Cre recombinase 60226 : sgRNA cloning backbone...

  14. Lentiviral Prep Service

    Type
    Collection
    ...pooled libraries 73178 Human sgRNA library Brunello in lentiGuide-Puro Human sgRNA library in backbone lentiGuide-Puro...Human CRISPRa sgRNA library Calabrese in backbone XPR_502 (P65 HSF), Set A Human CRISPRa sgRNA activation...containing 56,762 unique sgRNAs. This backbone does not contain dCas9. 56,762 unique sgRNAs targeting 18,885 ...pooled libraries 73633 Mouse sgRNA library Brie in lentiGuide-Puro Mouse sgRNA library in backbone lentiGuide-Puro...targeting 19,114 genes and containing 76,441 unique sgRNAs along with 1000 non-targeting controls. This backbone...backbone does not contain SpCas9. 76,441 unique sgRNAs targeting 19,114 human genes along with 1000 non-targeting...Human gRNA pooled library in lentiCRISPRv2 Human sgRNA library in backbone lentiCRISPRv2 targeting 19,114...

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

    Type
    Blog Post
    ...cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate flanking homology into a germline...don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want to work with gene drives? Then do...organisms but not wild organisms. For example, an sgRNA-only drive will spread exclusively through populations...express Cas9 from an unlinked locus, while a Cas9+sgRNA drive targeting a synthetic sequence will only spread...restore the wild-type sequence (save for the residual sgRNAs and possibly cas9 gene). An immunizing reversal...

  16. Hot Plasmids and Viral Preps - September 2021

    Type
    Blog Post
    ...Veening is now available with one sgRNA per operon or gene. The sgRNA pool can also target core operons... cell lines. The Turner Lab Human messenger-RBP sgRNA Library targets human mRNA binding proteins with...operons in other pneumococcal strains. The BARBEKO sgRNA library uses CRISPR cytosine base editors to disrupt...

  17. CRISPR Plasmids - Empty gRNA Vectors

    Type
    Collection
    ...pyogenes Joung sgRNA with U6 promoter 48962 Worm HindIII none S. pyogenes de Bono sgRNA with rpr-1 promoter...SpBsmBI sgRNA Hygro 62205 Mammalian/Lentiviral BsmBI none S. pyogenes Hygro Maehr pLenti SpBsmBI sgRNA Puro...dCas9-VP64 along with 1-4 sgRNAs expressed from independent promoters. sgRNAs are cloned into separate ... and allow for expression of more sgRNAs in a single vector. sgRNAs are expressed as polycistronic glycine...pLenti-sgRNA-Lib 53121 Mammalian/Lentiviral BsmBI none S. pyogenes ccdB Wei pLKO5.sgRNA.EFS.PAC 57825 ...Church LRG (Lenti_sgRNA_EFS_GFP) 65656 Mammalian/Lentiviral LIC none S. pyogenes GFP Vakoc U6>sgRNA(F+E) 59986... Boxem PU6::klp-12_sgRNA 46170 Worm PCR (see paper) none S. pyogenes Calarco SP6-sgRNA-scaffold 47912 ...

  18. Genomic Deletions in Mammalian Cell Lines

    Type
    Collection
    ...example, sgRNA-A’s protospacer sequence and PAM happen to fall on the bottom (Crick) strand while sgRNA-B’s...primers flanking each sgRNA target site (within 150 - 350 bp) to amplify the sgRNA target site to examine...complement: 1.0 μl sgRNA 24- or 25-mer oligo (100 μM; see step 1.4 ), 1.0 μl sgRNA 24- or 25-mer reverse...composed of a polyclonal population exposed to sgRNA-A and sgRNA-B (see step 5 ). The purpose of the following...delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a...posted at the top of the page. CRISPR Design Design sgRNAs manually or using freely available online tools...non-coding elements. For creating a gene knockout, two sgRNA located within exons will enrich even monoallelic...

  19. CRISPR Plasmids - Prime Edit

    Type
    Collection
    ...nicking sgRNA BbsI No Vermilion Norbert Perrimon 164423 pHSG1C3 Mammalian U6 pegRNA + nicking sgRNA BbsI ... RNA-H1-nick sgRNA-mCherry Mammalian, AAV hU6 + H1 pegRNA + nicking sgRNA No mCherry Hyongbum ...HindIII No Tilmann Weber 172717 pnsgRNA Bacteria J23119 (BBa_J23119) nicking sgRNA No Tilmann Weber 173220 pPBT-peRNA_GG-Puro...mutations in RT enzyme PE3 - PE2 plus additional sgRNA PE4 - PE2 plus MLH1 (a protein component of MMR)...the type of gRNA (e.g., pegRNA, epegRNA, nicking sgRNA), cloning enzyme, selectable marker, and whether...David Liu 140448 QPM-sgR (pTaU3) Plant TaU3 nicking sgRNA Eps3I + NcoI No Caixia Gao 141081 pYPQ141D-peg Plant...BbsI for sgRNAs; BbsI + PstI for pegRNAs No Xiao Wang 170132 pOsU3 Plant OsU3 pegRNA BsaI + HindIII No Caixia...

  20. What's New in CRISPR - September 2019

    Type
    Blog Post
    ...targeting requires two sgRNAS: one that targets the knock-in site and a universal sgRNA that targets the donor... the genomic loci of interest. As the universal sgRNA is not predicted to target sites in the human, pig...
Showing: 1 - 20 of 94 results