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We narrowed to 304 results for: cas9

Showing: 1 - 20 of 304 results

  1. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast... express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast was...additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts...

  2. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    ...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences... target effects of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and...non-target strand and Cas9, Kleinstiver et al disrupted interactions between Cas9 and the phophate backbone...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target...portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and SpCas9-HF1 each... and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker...your Cas9 homolog of choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver...

  3. CRISPR References and Information

    Type
    Collection
    ...sites for S. pyogenes Cas9, S. aureus Cas9, S. thermophilus Cas9, N. meningitidis Cas9, or Cas12a from your...vectors using ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H Cas9 ; p201N Cas9 ; pUC gRNA Shuttle PDF, 500 KB...CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or Cas9 D10A (pX335) PDF,...118 KB Church gRNA design and cloning for Cas9 orthologs Cas9 plasmids PDF, 107 KB Chen and Wente Zebrafish...vitro transcription, injection gRNA core ; Cas9 ; optimized Cas9 PDF, 66.8 KB Fujii gRNA design and cloning... Injection and selection for Cas9-triggered homologous recombination Cas9-sgRNA target construct ; pMA122...destination: pAC90 PDF, 1.1 MB Katic Nematode: Cas9 and gRNA use Cas9 (pIK86) ; gRNA empty backbone (pDR274) ...

  4. Components of CRISPR/Cas9

    Type
    Blog Post
    ...sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants...basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related...the PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...genomic target for Cas9, and the tracrRNA which acts as a scaffold linking the crRNA to Cas9 and facilitates... to direct Cas9 to a specific genomic locus and the scaffolding sequence necessary for Cas9 binding. When...CRISPR/Cas9 for genome editing, researchers simply need to express a gRNA designed to direct Cas9 to their...tracrRNA and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound to the Cas9/tracrRNA...

  5. Zhang Lab CRISPR Page

    Type
    Collection
    ...Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR/Cas9 Synergistic...of cells and nuclei. Return to top AAV - Cas9 mouse CRISPR-Cas9 is a versatile genome editing technology...constitutively expressing Cas9 knockin mice (Platt et al ., Cell 2014). In these mice the CRISPR-Cas9 system can be...constitutively Cas9-expressing mouse. Described here are AAV vectors that can be combined with Cas9 in a wide...are below. The Cas9 knockin mouse can be purchased from Jackson Labs: Cre-dependent Cas9 mouse Constitutively...cloning information: CRISPR-Cas9 mouse toolbox protocol 637.8 KB CRISPR-Cas9 Cre expression vectors for... (20bp). CRISPR-Cas9 Cre expression vectors for genome editing in the brain Using Cas9 mice Platt et al...

  6. CRISPR Guide

    Type
    Collection
    ... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...strand SpCas9-HF1 — disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 — increase Cas9 proofreading...more Cas9 fusion tools later in the guide. Figure 3: Overview of Cas9n. Increasing fidelity of Cas9 Researchers...enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no defined...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...constructs with Cas9 fused to proteins that modulate HDR or NHEJ components, like miCas9 , Cas9-DN1S , or others...Plasmids: Purify Cas9 Alternatives for CRISPR Genome Engineering S. pyogenes Cas9 (SpCas9) is the most commonly...

  7. CRISPR Plasmids - Empty gRNA Vectors

    Type
    Collection
    ... plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that can cut, activate...Expression System Cloning Enzyme Co-expressed Cas9 Cas9 System Selection PI pCRISPR 42875 Bacteria BsaI...BbsI)_CBh-Cas9-T2A-BFP 64323 Mammalian BbsI yes, cut S. pyogenes BFP Kuhn pU6-(BbsI)_CBh-Cas9-T2A-mCherry...meningitidis Joung Cas9 sgRNA vector 68463 Mammalian U6 none S. pyogenes Zhang Cas9/pTREX-n 68708 Other...independent U6 promoters and Cas9 from CBh promoter. Ventura A CRISPR/Cas9 toolkit for multiplex genome...cassettes are assembled into a Cas9 expressing vector. Yang A Robust CRISPR/Cas9 System for Convenient, High-Efficiency...accessory pack is available for dCas9 and FokI-dCas9. Yamamoto Multiplex CRISPR/Cas9-based genome engineering...

  8. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...assessment of the off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect...the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants that were able...consideration when choosing a Cas9 is balancing specificity and sensitivity. Wild-type Cas9 exhibits high on-target... directed evolution to generate Sniper-Cas9 resulting in a Cas9 with improved specificity. They compared...on-site and off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths...loci and gRNA lengths. Recent alternatives to Cas9 While Cas9 remains the most widely used CRISPR nuclease...

  9. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ... can occur due to prolonged Cas9 expression     The requirement for Cas9 transcription and translation...consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required and...express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier’s ... of Cas9-gRNA RNPs by specific cell types (Rouet et al. 2018). This was accomplished using a Cas9 mutant...for experiments where stable Cas9 expression is required, such as when a Cas9 fusion to a fluorescent protein...and stability of the Cas9 protein in the host. Indeed, research has shown that a Cas9 protein from Geobacillus...

  10. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ... gRNA, Cas9 binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators... a potent Cas9-based transcriptional regulator without making any functional changes to Cas9. For instance...repression with nuclease-competent Cas9. When complexed with a truncated gRNA, Cas9 is still able to bind DNA ...tight control over Cas9 nuclease activity while eliminating the reliance on orthogonal Cas9 species. Within...capability, and this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains...importantly, most Cas9 orthologs are less well characterized than the conventionally used SpCas9 (derived from...nuclease-competent Cas9, obviating the need to generate new cell lines or transgenic animals expressing dCas9 as a ...

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

    Type
    Blog Post
    ...proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing ...heterodimerizing domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure...modular nature of Cas9, Nihongaki et al. showed that it was possible to swap out the Cas9 domains in their...light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription...p65 and VP64 which in turn transcribes N-Cas9. The N- and C-Cas9 fragments then dimerize to form a functional...Off Genome Editing Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved using single-chain...

  12. CRISPR History and Development for Genome Engineering

    Type
    Collection
    ...High fidelity Cas9s: Three high fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very ...III and Cas9 enzymes, which together separate the individual crRNAs. (3) The tracrRNA and Cas9 nuclease...Staphylococcus aureus (SaCas9) is smaller than SpCas9 and more easily packaged in AAV. Cas9 orthologs also have...beyond SpCas9, moving us closer to being able to target every locus in every genome. Multiple Cas9 orthologs...capability of Cas9 to make other types of modifications at specific loci. Activate or Repress : dCas9 fused to...the crRNA would bind. (5) After the complex binds, Cas9 separates the double stranded DNA target and cleaves...Single multi-domain effector) Type I (Cas3) Type II (Cas9) Type III (Cas10) Type V (Cpf1) Type IV (Csf1) Type...

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

    Type
    Blog Post
    ...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy...Components of CRISPR  xCas9: Engineering a CRISPR Variant with PAM Flexibility Cas9 vs the Other Cases Further... with a nearby protospacer-adjacent motif (PAM)? Cas9 is becoming an ever more ubiquitous tool for genome... bridge helix. Created with BioRender.com.   Cas9 has two major lobes, called NUC (for nuclease) and...system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs to, and their...

  14. Lentiviral Prep Service

    Type
    Collection
    ...Root Cas9 Lentivirus Cas9 viruses can be used to interrogate the genome in a variety of ways. Cas9 proteins... 52962 lentiCas9-Blast Sp Cas9 Cut Blasticidin Expresses human codon-optimized S. pyogenes Cas9 protein...backbone. Zhang Cas9 and Accessories for Activating Gene Expression Catalytically-dead Cas9 (dCas9) can be fused...plasmids resource page . Cas9 for Editing Genomic Sequences To perform genome edits, Cas9 nuclease is used to...Browse In-Stock Lentivirus Pooled CRISPR Libraries Cas9 Pooled Barcoding Libraries Control Addgene's lentiviruses...Resistance Activity PI 61422 dCAS9-VP64_GFP dCAS9 (D10A, H840A) none Expresses dCAS9-VP64 activator with 2A ...-targeting controls. This backbone contains SpCas9. SpCas9 and 76,441 unique sgRNAs targeting 19,114 human...

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

    Type
    Blog Post
    ...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific... CRISPR/Cas9 has made it easier than ever to make precise, targeted genome modifications. Cas9 has been...introducing Cas9-mediated frameshift mutations in the gene of interest. The genomic target for Cas9 and the... system. This system uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has...targeted to the promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other SAM factors...Next CRISPR Experiment Cas9 expression systems Addgene carries a variety of Cas9 containing plasmids that...target two Cas9-nickases (Cas9n) to the target locus, enhancing specificity over a single SpCas9. The drawback...

  16. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...required for Cas9-based activation. CRISPRa for genome-wide studies One of the best uses for Cas9 activators...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background...there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow...upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a transcriptional...

  17. Worm Expression Resources

    Type
    Collection
    ... via a CRISPR-Cas9 system. Cloning-free CRISPR for C. elegans , which uses purified Cas9, such as that...feeding worms bacteria expressing dsRNA or use CRISPR/Cas9 gene editing to easily engineer specific transgenic...worm CRISPR resources include plasmids expressing Cas9, empty gRNA backbones, and constructs for endogenous...targeting vectors for tag insertion using CRISPR/Cas9 and the selection of C. elegans strains. CRISPR ...that expressed from pHO4d-Cas9 from Michael Nonet. Protocol can be found at the (Link opens in a new window...Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. Mos1 Based Transgenesis...Blog Posts An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency Fluorescent Tagging of Endogenous...

  18. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    ...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs... aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced...truncated Cas9. Unfortunately, certain drawbacks limit the utility of these systems: St1Cas9 requires ...used CjCas9 with AAV to target genes in mouse muscle and eye tissue. Neisseria meningitidis Cas9 (NmeCas9...

  19. Introducing an All-in-One CRISPR/Cas9 Vector System for Multiplex Genome Engineering

    Type
    Blog Post
    ...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our...all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available...one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma...offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ... based on earlier evidence showing that a CRISPR/Cas9 plasmid could be injected directly into mice for...

  20. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting... Cas9 delivery Overview: Before any editing can begin we need our A549 cells to express Cas9. In these...with too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (... variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression...to assay Cas9 activity in my stable A549 cell pools. Using this virus, it’s expected that Cas9 activity...gene. So, cells with no Cas9 should appear green and cells with functional Cas9 should appear less green...
Showing: 1 - 20 of 304 results