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We narrowed to 609 results for: CaS;

Showing: 151 - 200 of 609 results

  1. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed PMID...anaerobic conditions needs to be examined on a case-by-case basis. GFP is sensitive to acid The chromophore...nuclease-deficient Cas9 can bind loci in the genome that are defined by a gRNA. Since Cas9 is a protein, a...the localization of the fusion protein. Also, in cases where the protein of interest is relatively small...the function of the protein. This is clearly the case when proteins undergo post-translational modification...

  2. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assembly of DNA molecules...modularity to overlap-directed methods (Casini 2014). In this case linker sequences are added before the...In this case, rather than using a restriction enzyme or PCR to make linearized vector, the Cas9 enzyme ...standard sequences with overlap regions, such as in the case of MODAL (modular overlap-directed assembly with...restriction site for linearization. This is a perfect case for the use of Gibson assembly combined with the...assembled into a vector using Gibson assembly. In this case the vector was PCR amplified to contain regions ...Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly ...

  3. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    Published
    April 5, 2018, 12:42 p.m.
    ...carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering." Cell 154 (2013):1370... 47327 pET-28b-Cas9-His 6xHis (C terminal); NLS (C terminal) 62933 pET-Cas9-NLS-6xHis 6xHis.... recommend ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components (..., we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et al. (2018) suggest...insertion site to maximize HDR efficiency. In this case, we’re fortunate that there’s a guide that directs... and cleavage sites (slashes) are shown. In this case, the green guide would be selected, since it cleaves...increase T7 promoter activity. To avoid re-cutting by Cas9 after editing, make sure that the repair template...

  5. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    ...allow positively charged ions such as Na+, K+, and Ca++ to passively enter the cell with the concentration...function through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation...control GPCR activation and downstream signaling cascades (reviewed in Abreu & Levitz, 2020), including ...extracellular domain which activates the native signaling cascade of the intracellular part of the receptor, similar... out important branch points along a signaling cascade that may go awry in a disease state.      ...

  6. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    Published
    July 6, 2021, 1:28 p.m.
    ...in Vibrio cholerae that encodes a Type I-F CRISPR-Cas system. This system has four major components: 1)...crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that forms the QCascade DNA-targeting module with the ... transposons encode reporter genes, mutagenic cassettes, or barcodes. When transposons are delivered to...For the INTEGRATE system to transpose, first the QCascade complex surveys the genome and binds its target...molecule, and inserts it ~50 bp downstream of QCascade’s genomic target site (Fig. 3B). The INTEGRATE ...of RNA-guided transposition with INTEGRATE. The QCascade complex surveys the genome and binds its target...molecule, and inserts it ~50 bp downstream of QCascade’s genomic target site.  T-RL= transposon right-...

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

    Type
    Blog Post
    Published
    Oct. 24, 2017, 1:54 p.m.
    ...Find out more at www.projbridge.com. Walnut Creek, CA, USA 37.9101°N 122.0652°W – Steph Hays When I marched...22 to join in the March for Science.  Cold and overcast with intermittent rains, it was weather befitting...

  8. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...Addgene. 10. Port, Fillip, et al. “Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering...humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric...options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator...including Standard PolIII-gRNA cassette, Csy4-cleavable cassette, and PTG cassette.    Find gRNA multiplexing... then digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination ... encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 variants are available:...the multiple gRNAs and Cas9 together into a single plasmid. Although only wt hCas9 entry vectors are supplied...

  9. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...either Neo or Hygro resistance markers with CRISPR/Cas vectors targeting their genes of interest to generate...CRISPR-Cas9-based tool for targeting CpG methylation by fusing catalytically inactive Cas9 (dCas9) with...pDGE Dicot Genome Editing Kit contains Cas9, Cas9 nickase, and Cas9 activator expressing plasmids that can...high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans CRISPR/Cas9 technology has revolutionized...reported on a novel Cas9-based activator developed by the Church lab, termed “dCas9-VPR”, which facilitates...creating a new dCas9-VPR expression plasmid that uses the piggybac transposase to integrate dCas9-VPR into ... is genomic integration of dCas9-VPR and controlled expression of the dCas9-VPR transgene by doxycycline...

  10. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ... potential applications. Polstein LR & Gersbach CA, Nat Chem Biol 2015 Mar;11(3):198-200. SunTag system...fully functional Cas9 upon dimerization. Splitting wild-type Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal...specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES). This...gene activation when fused to dCas9. In the dCas9-SunTag-VP64 system, dCas9 is fused to a scaffold containing...domains onto dCas9 to generate a dCas9-VP64-p65-Rta fusion protein (referred to as dCas9-VPR) had a synergistic... can write a blog post about it! Split Cas9 system The Cas9 protein is composed of an n-terminal DNA... the bi-lobed architecture of Cas9 to engineer a series of "split" cas9 molecules that cannot function...

  11. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    Published
    Jan. 10, 2017, 3:37 p.m.
    ... gRNA, Cas9 binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators...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...to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative nuclease... 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 ...on rare occasions truncated gRNAs (≤15nt) have been found to retain some ability to induce Cas9 to make...

  12. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    Published
    Sept. 6, 2018, 12:02 p.m.
    ... 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...long-term expression of Cas9, such as visualizing genomic loci using fluorophore tagged dCas9 may require the ...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...

  13. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...The popular CRISPR base editing technique uses dCas9/Cas9 nickase fused to a cytidine deaminase for targeted..., a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently tested the original BE3...Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169  pJL-SaBE3  SaCas9 (NNGRRT)    85170  pJL-SaKKH-BE3... pJL-SaKKH-BE3  SaCas9 (NNGRRT)    85171  pBK-VQR-BE3  VQR-Cas9 (NGA)  Lower off-target activity than...-BE3  EQR-Cas9 (NGAG)  Lower off-target activity than BE3  85173  pBK-VRER-BE3  VRER-Cas9 (NGCG)   ...85174  pBK-YE1-BE3  SpCas9 (NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window...85176  pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target...

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

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...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...

  15. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    Published
    Jan. 8, 2019, 1:54 p.m.
    ...Addgene!) pX330-U6-Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome... with lentiCas9-Blast or with cell lines already expressing Cas9. Find lentiGuide-Puro. pSpCas9n(BB)-2A-GFP... Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses both a Cas9 protein and...Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus... sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP...chimeric guide RNA. Find pX330-U6-Chimeric_BB-CBh-hSpCas9. pSpCas9(BB)-2A-Puro (PX459) V2.0 - This plasmid from... in the puromycin cassette that makes it less efficient in some cell lines. pSpCas9(BB)-2A-Puro (PX459...

  16. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ...or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 cleavage at predicted...expression of Cas9/gRNAs, which could result in higher rates of off-target cleavage. In both cases, translating...were located near the Cas9 cut site, leading Wang et al. to conclude that some Cas9-derived indels may not...CRISPR/Cas9 escape occurs in an animal model. If these escape mutations are frequent, the dCas9-mediated...strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy ...destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read ...realistic with the advent of CRISPR. Using CRISPR/Cas9 SAM to "shock and kill" To “shock and kill” the...

  17. Top Requested Lentivirus and AAV of 2016

    Type
    Blog Post
    Published
    Jan. 6, 2017, 3:56 p.m.
    ... expressed from lentiCas9-Blast. Purpose of LentiCas9-blast LentiCas9-blast is a Cas9 expression vector...express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be used to generate Cas9-expressing...While various Cas9 mutants have been engineered to perform unique editing tasks, classic Cas9 nuclease, which...Secondly, you can use a LentiCas9-Blast viral prep to create a single stable Cas9-expressing cell line and...gRNAs in a monoclonal Cas9 background because you’ll have more consistent levels of Cas9 expression between...this plasmid Cas9 expression is typically cytotoxic, which makes generation of high titer Cas9 lentiviruses...growth rate variability when using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this...

  18. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    Published
    March 24, 2016, 2:30 p.m.
    ...Postdoctoral Researcher in Jacob Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts...replacement by HDR. Understanding Cas9 biophysics enhances HDR We first measured Cas9 dissociation rates and discovered...interaction with the Cas9-DNA duplex. Image from Richardson et al., 2016.   dCas9 enables sequence replacement...normally accompanies Cas9 cutting. We still don’t know the mechanism underlying dCas9 editing, but it could... cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors...factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay... and thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining...

  19. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    Published
    May 30, 2019, 12:44 p.m.
    ...catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific...targeting gRNA. But dCas9 does not cleave the DNA. Much of the research using dCas9 has focused on transcriptional...mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting their...editing, numerous discoveries have been made using the Cas9 nuclease in the absence of genomic alterations. ...activators (Qi et al., 2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a...genomic region using the CRISPR system, consisting of dCas9 and targeting gRNA, to identify molecular interactions...3xFLAG tag, allowed these researchers to isolate dCas9 with the bound genomic locus, which they then confirmed...

  20. Your Top Requested Plasmid in 2016!

    Type
    Blog Post
    Published
    Jan. 11, 2017, 8:47 p.m.
    ... un-tagged versions of SpCas9 and further variants replace SpCas9 with SpCas9n (nickase), which nicks ... express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid ...tools came out including the single base editors, Casilio, CombiGEM, and a variety of pooled libraries. Not... as is obvious from our most requested plasmid, SpCas9 is still going strong as the basis for many genome...further ado, the most request plasmid in 2016 was... pSpCas9(BB)-2A-Puro (PX459) V2.0       Similar to last...2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang Lab. It's name, though a little ...plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified version of CAG promoter...

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

    Type
    Blog Post
    Published
    Dec. 20, 2017, 2:33 p.m.
    ...Careers and Rachel Casseus, Esq. Founder of Casseus Law. Rachel Casseus Esq., Founder Casseus Law. Driven by...1A and EB-1B cases, Casseus Law recommends 6-8 letters.  For these recommendations, Casseus Law suggests...Celano of Propel Careers and Rachel Casseus, Esq. Founder of Casseus Law. Disclaimer: The contents of this...learn more, visit www.propelcareers.com. Casseus Law: Casseus Law is dedicated to the practice of US immigration...law, Casseus Law provides legal services to people located in all 50 states and globally. Casseus Law ...uncertainty associated with filing a permanent residence case with United States Citizenship and Immigration Services...obtain permanent residence. The recently established case Matter of Dhanasar provides the three-prong test...

  22. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    Published
    March 19, 2019, 1:31 p.m.
    ...great about CASANOVA? It works with the S. pyogenes Cas9, dCas9-effector fusions, and xCas9, and requires... CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  Listen to the anti-CRISPR podcast segment...potent Acr-LOV fusion protein inhibitor CASANOVA for “CRISPR-Cas9 activity switching via a novel optogenetic...AcrIIA4.” CASANOVA responds to blue light quickly. Within minutes of inactivating the CASANOVA complex,...CRISPR systems including the popular S. pyogenes Cas9 (Rauch et al., 2017, Hynes et al., 2017). However..., they have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et al., 2018)...., inhibiting function of Acr. This allows CRISPR-Cas9 to function on its target.  They name their most...

  23. What's New in CRISPR - May 2019

    Type
    Blog Post
    Published
    May 28, 2019, 12:49 p.m.
    ... AAV delivery of Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida albicans...paper in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted ...After testing dCas9 fusions with transcriptional repressors, they found that fusing dCas9 to an Mxi1 repressor...gene. Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a new CRISPR tool...library Covalent tethering of DNA repair template to Cas9 Homology-directed repair (HDR) can be used to make...oligodeoxynucleotide HDR template covalently attached to the Cas9-gRNA RNP complex via the fused HUH endonuclease ...

  24. Hot Plasmids - September 2019 - CRISPR, Auxin Inducible Degradation, MerMAID Channelrhodopsins, and Genome Looping

    Type
    Blog Post
    Published
    Sept. 3, 2019, 1:15 p.m.
    ...range of CRISPRCas9 by engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9 is widely...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...recognize the target. Osamu Nureki’s lab engineered SpCas9 (SpCas9-NG) with relaxed preferences for the third ...NGG. In addition to the engineered SpCas9, they also fused the SpCas9-NG with the activation-induced cytidine...  The Kanemaki lab previously developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116...widely used genome editing tool. However, Cas9’s endonuclease activity can be limited by the need for a specific...cells. The increased versatility of the engineered SpCas9-NG and its variants can be used to engineer specific...

  25. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    Published
    March 21, 2019, 12:47 p.m.
    ...different Cas12a inhibitors. Some of these anti-Cas12a proteins have broad inhibitory effects on Cas12a orthologs... research.     CRISPR-Cas12b for human genome editing The best studied Cas12b nuclease from Alicyclobacillus...The Feng Zhang lab identified a Cas12b from Bacillus hisashii, BhCas12b that is functional at 37 °C but...Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion... - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated...Alicyclobacillus acidoterrestris (AacCas12b) exhibits optimal DNA cleavage at 48 °C, a temperature incompatible...tRNA adenosine deaminase that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits...

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

    Type
    Blog Post
    Published
    Oct. 4, 2018, 12:44 p.m.
    ...combining specific sgRNAs with Cas9 or Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks... loss of function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or ...an inactive RNA-guided DNA binding protein (dCas9). dCas9 is then tethered to a repressive domain, such... issues. CRISPRi libraries utilize an engineered Cas9, which contains multiple point mutations in the ...library In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation....developed to recruit transcriptional machinery to dCas9 and its RNA directed binding site. These methods... fusion of an activation domain such as VP16 to dCas9 or the recruitment of multiple copies of VP16 termed...

  27. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    Published
    Feb. 21, 2019, 1:15 p.m.
    ...nicking Cas9. A guide directs Cas9 to a site of interest, where it nicks its target DNA (left). Cas9 dissociates...enhance nCas9 (enCas9) EvolvR with a targeted mutagenesis rate ~9-fold higher than the original nCas9, while...error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar to mutagenesis with error prone PCR...used to direct the PolI3M-nCas9 complex to a DNA site of interest, which nCas9 nicks and then dissociates...amino acid changes were introduced to nCas9. These changes promote nCas9’s dissociation from DNA after nicking... a user-defined genomic region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the...diversification by targeting a site of interest with dCas9 fused to the Activation Induced Cytidine Deaminase...

  28. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    Published
    Sept. 26, 2019, 12:50 p.m.
    ...releasing Cas9. Nanoblades can also deliver Cas9 variants, such as a transcriptional activator dCas9-VPR, ...centrifugation. Gag-Cas9 fusion protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated ...Nanoblades by association with the the Cas9 portion of the Gag-Cas9 fusion protein. A single Nanoblade particle...along with the gRNA and Cas9. An “all-in-one” Nanoblades which carries a gRNA, Cas9, and a HDR donor template... leukemia virus (MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the...generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery vehicle that’s based on mouse leukemia...just like the unmodified MLV Gag protein. After Gag-Cas9 is incorporated, the MLV protease, supplied as part...

  29. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    Published
    Sept. 24, 2015, 2:30 p.m.
    ... CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts...in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro transcription...generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro transcribed... mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator or repressor...need to deliver. Minimally, one or more sgRNAs and Cas9 are required for any application. If you want to...Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible. U6 ...stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a mammalian cell line that can be...

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

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ...prime editor consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the... that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9 treatment causes...ways. In some cases, however, combining an evolved RT domain from PE6a-d with an evolved Cas9 domain from... ~ ~ ✓ ✓ ✓ R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ...homology to the genomic DNA locus. After nicking by Cas9, the homologous pegRNA sequence hybridizes to the...-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1). But innovations by the Liu ... make a specific edit defined by the pegRNA. In cases when bystander editing is unacceptable, prime editors...

  31. Hot Plasmids: Spring 2025

    Type
    Blog Post
    Published
    June 3, 2025, 1:15 p.m.
    ...-for-one solution: TurboCas!TurboCas provides the genomic targeting power of dCas9 with the dynamic labeling... While dCas9 has been used in combination with proximity labeling enzymes before, such as CasID, TurboCas...background labeling. By fusing miniTurboID with dCas9 to create TurboCas, the Shilatifard Lab created a very quick...editing with STITCHR Dynamic protein labeling with TurboCas Enhancer-AAV viral preps targeting interneurons...Bentley For RNA-guided genome editing, think beyond Cas9 with STITCHR: a combined CRISPR / retrotransposon... Figure 4: STITCHR consists of a nicking Cas9 fused to a shortened R2Tocc transposon protein, ...for scarless insertion of the payload (teal). The Cas9 gRNA targeting guide and the transposon RNA homology...

  32. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...editors use either catalytically dead Cas9 (dCas9) or a Cas9 nickase (nCas9) to avoid introducing double-strand...need from Cas9 are sequence specificity and the R-loop structure. In order to bind DNA, Cas9 requires ...non-target strand of DNA for Cas9 to bind.   R-loop The structure formed by Cas9 binding to DNA and separating...advantage of repair pathways within the cell — in some cases, the same repair pathways that were considered problematic...PAM) on the opposite strand. Once it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate...single-stranded DNA (ssDNA) region, using enzymes fused to Cas9 to alter the exposed ssDNA bases. Thus, base editors...window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR, the non-target strand...

  33. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    Published
    Aug. 18, 2015, 2:30 p.m.
    ...CRISPR/Cas9 over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists...in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be ...of two components: the non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20...the user, and it can be easily modified to target Cas9 to virtually any genomic locus, provided the target...adjacent motif (PAM) sequence. Co-delivery of wild-type Cas9 and a gRNA generates a double-strand break in the...genomic target. The goal of a CRISPR screen is to use Cas9 and a pool of gRNAs to identify genes that are essential...lentivirus to deliver a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral CRISPR libraries...

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

    Type
    Blog Post
    Published
    May 21, 2019, 12:10 p.m.
    ...activate a cascade of caspases, protease enzymes that cleave proteins. Executioner caspases are activated... executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase cleavage site...one of the linkers is a consensus caspase-3 (an executioner caspase) cleavage site (DEVD). Upon cleavage...activated last in the cascade and are responsible for the degradation of over 600 cellular components ultimately...used genetically encoded fluorescent executioner caspase reporters. Several apoptosis reporters have been...site. Cutting the linker by a caspase results in a change in fluorescence emission. FRET and fluorescence...Studying apoptosis in vivo using the FlipGFP-based caspase reporter The Shu Lab successfully used FlipGFP ...

  35. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    Published
    Aug. 4, 2016, 2:30 p.m.
    ... preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids. Cas9 guide RNA delivery...Targeting Specificity with eSpCas9 and SpCas9-HF1 See How Cpf1 Stacks Up to Cas9 and NgAgo Resources on ...genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering methods...engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss ...head-to-head.  Exciting features of NgAgo First, unlike Cas9, NgAgo does not require a PAM sequence for target... guide sequence rather than an RNA guide as with Cas9. Third, it can be used to target “difficult” GC-...the other hand, the widespread success of CRISPR-Cas9 across multiple model organisms (3) lies in the ...

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

    Type
    Blog Post
    Published
    June 27, 2018, 1:16 p.m.
    ...genomic locus: Caspex  Article contributed by Susanna Bachle  Listen to the Caspex podcast segment Steven...lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9) fused to APEX2 is expressed from a Tet/Shield1...please sign up here. Listen to the Hot Plasmids Podcast!  MarathonRT: A highly processive reverse transcriptase...contributed by Will Arnold  Listen to the MarathonRT podcast segment Researchers from Dr. Anna Pyle’s lab at...Marcy Patrick  Listen to the nanobody toolkit podcast segment Nanobodies are single domain antibody fragments...genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered peroxidase, (APEX)... proteins with biotin as an affinity tag. This ‘Caspex’ construct is guided by a pre-defined sgRNA sequence...

  37. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    Published
    July 2, 2020, 12:40 p.m.
    ...co-expressed, the gRNA targets the dCas9 to the loci of interest and dCas9 is biotinylated by BirA. From here...To increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ... cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA, a biotin...be pulled down via streptavidin IP. Biotinylated dCas9 goes along for the ride throughout all of these ... CAPTURE cell lines will have varying levels of dCas9 and gRNA expression. Additionally, CAPTURE 3C-based...CAPTURE 2.0:  Replace the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long...three to two. Lentiviral delivery of BioTAP-tagged dCas9 and gRNAs. Using lentiviral delivery of the two ...

  38. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ...summarize, CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of transcriptional...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...limited to in vitro settings due to the large size of Cas9 activators, which largely precludes delivery by ...mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up to twenty copies of the... transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More...packaging limits (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH...here. Neuron-optimized viral vectors Alternatively, Cas9 activators can be delivered to the CNS by lentivirus...

  39. New CRISPR Web Resources and #12DaysofCRISPR Recap

    Type
    Blog Post
    Published
    Dec. 12, 2017, 3:13 p.m.
    ... CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation:...contains Cas13 constructs for mammalian and bacterial systems. Plasmids with wild-type Cas13a (formerly...RNA cleavage in bacteria. In mammalian systems, Cas13a degrades only the specified RNA target. Bonus: ...This characteristic probably explains why pC016 - LwCas13a guide expression backbone with U6 promoter has...CRISPR? I certainly didn’t, but now we can with Cas13b/ADAR constructs from the REPAIR (RNA Editing for...and CRISPR Antimicrobials Day 7: Multiplexing and Cas9 variants: CRISPR 101: Multiplex Expression of gRNAs...The PAM Requirement and Expanding CRISPR Beyond SpCas9 Day 8: Repair mechanisms: CRISPR 101: Non-homologous...

  40. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    Published
    Nov. 3, 2022, 12:15 p.m.
    ... eSpCas9(1.1), or HypaCas9. Read more about Cas9 options in our blog post here. Dealing with low editing...version of Cas9 that has been specifically engineered for reduced off target editing, such as SpCas9-HF1, eSpCas9...gRNA and introduced it into your target cells with Cas9. Hooray! Now it’s time to make sure your genome ... product of both an unedited population and your Cas9-targeted cells. Simply upload both trace files to...with the estimated editing frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure ...introduced a donor template along with your gRNA and Cas9 combo and are hoping for homology-directed repair..., with only a few base pairs modified? In these cases, it’s best to do some planning in the design phase...

  41. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ...containing Cas9 and the PITCh-gRNA. Step 3: Contransfect the PITCh vector with the vector carrying Cas9 and ...protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of a stop codon...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary for...for knock-in: one on either side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies...microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus (see figure below). The double-...of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA...plasmic is cotransfected with a plasmid carrying Cas9, the PITCh-gRNA, and the locus-specifc gRNA. The...

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

    Type
    Blog Post
    Published
    Sept. 18, 2018, 1:33 p.m.
    ...Harris lab showed that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more ...contributed by Klaus Wanisch  Listen to the optogenetics podcast segment LOVTRAP is an optogenetic system that ...contributed by Tyler Ford  Listen to the riboglow podcast segment Researchers from the Palmer lab at the ...contributed by Caroline LaManna  Listen to the mCarmine podcast segment Scientists are always on the lookout for...reporter for rapid quantification of base-editing and Cas9 cleavage activity Article contributed by Angela ...Angela Kaczmarczyk  Listen to the base editing podcast segment The increasing popularity of single base-editors...fluorescence-based quantification of base editors that utilize dCas9 fused to cytidine deaminases like APOBEC. To achieve...

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

    Type
    Blog Post
    Published
    April 7, 2015, 4:21 p.m.
    ...) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease activity coupled.... Using dCas9 as an on/off switch The multiple scRNAs all individually utilize the same dCas9 protein....modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as a master...an orthogonal dCas9 protein that will still recognize guide RNA but with a different dCas9 binding module...specific proteins via aptamer domains. Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent...layered with distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field...applied to effectively change metabolic flux. The dCas9 protein and orthogonal variants can be used as regulatory...

  44. Healthcare Consulting: A Door to the Business of Life Sciences

    Type
    Blog Post
    Published
    Oct. 13, 2016, 2:30 p.m.
    ...consultants as short-term experts to work on “cases”. Cases present specific operational or commercial challenges...performance, and occasionally being faced with “shifting goal posts” as a part of a case, consulting can...include efforts like flexible staffing on case teams, where case managers are encouraged to use the wider...clients face imminently. Consulting firms deploy case teams, usually with 3-6 consultants, who work on...facing a crunch, and using lessons from previous cases to better staff future teams. As with any highly...become cauldrons on the verge of boiling. On other occasions, clients can be excessively demanding. For instance... critical analyses skills were applicable to my cases. I can directly use my understanding of the molecular...

  45. Unique and persistent IDs for improved reproducibility: Addgene now supports RRIDs and compact identifiers for all plasmids

    Type
    Blog Post
    Published
    Nov. 20, 2018, 8:17 p.m.
    ...lenti-Cas9-VQR plasmid page. Despite their differences, both of these PID formats for lenti-Cas9-VQR contain...resolvable compact identifier for every plasmid. In case these PID formats are new to you, we’ll go through...available through Addgene. Let’s take a look at lenti-Cas9-VQR-Blast, which has the Addgene ID #87155. The ...The resolvable compact identifier format for lenti-Cas9-VQR is the URL “http://n2t.net/addgene:87155” where...n2t.net web address. For the RRID format, lenti-Cas9-VQR is represented as “RRID:Addgene_87155” and the...formats. Let’s go back to our example plasmid, lenti-Cas9-VQR-Blast. If you scroll down to the bottom of this...semi-colons. For our example plasmid it will read “lenti-Cas9-VQR-Blast was a gift from Daniel Bauer (Addgene ...

  46. DAP arrays for multiplex gene editing

    Type
    Blog Post
    Published
    July 7, 2022, 1:15 p.m.
    ... found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...first thought to enable MBE by using Cas12a fused base editors (dCas12a-BE). However, the poor editing efficiencies...also exhibited reduced Cas9-dependent off-target editing and do not cause higher Cas9-independent off-target...only minimal payloads. DAP array  Although CRISPR-Cas12a, with abilities to process and release its own ...leading the edge of the precision gene editing. CRISPR-Cas9, the key component to enable efficient base editing...would independently generate multiple gRNAs for nCas9-BE, or any other kinds of CRISPR tools, without ...organisms and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array encoding...

  47. Hot Plasmids - May 2022

    Type
    Blog Post
    Published
    May 26, 2022, 1:15 p.m.
    ...inactive dCas9 and microbial SSAPs (dCas9-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP...cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et al, 2022.  Find dCas9-SSAP...Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced error rate for long sequence knock-ins... https://doi.org/10.1038/s41593-021-00969-4.   dCas9-SSAP with reduced error rate for long sequence knock-ins...Wilson Le Cong’s lab has developed a method using dCas9-SSAP to knock-in long sequences with reduced risks...of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large inserts. However, this system ...in comparison to the 62-90% error rate seen with Cas9 editors. The lab believes that this system could...

  48. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    Published
    April 6, 2017, 1:02 p.m.
    ...interest. To get the molecular components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the...accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures...regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise ...We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with...process following the double strand break CRISPR/Cas9 makes at the target site. The whole segment—a GFP...the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection. Making the double strand break...the fluorescent tag to the cells, we used CRISPR/Cas9 to make a precise cut in the genome near either ...

  49. What's New in CRISPR - December 2019

    Type
    Blog Post
    Published
    Dec. 17, 2019, 2:37 p.m.
    ...fusing catalytically inactivated RNA-targeting Cas13 (dCas13) with the adenine deaminase domain of ADAR2.... as these recent blog posts on prime editing, IgnaviCas9, and Nanoblades), but we hope that this blog ...toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase... a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-ACE (adenine and...Read the paper in bioRxiv Find Target-ACE   CRISPR/Cas9-based toolkit for epitope tagging in neurons Harold... the paper in bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery by lentiviral capsid-based...capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced off-target ...

  50. SciComm with the Experts at Science in the News Part 2

    Type
    Blog Post
    Published
    April 27, 2017, 2:30 p.m.
    ...Pint series. Chapter 4: The SITN Podcast, Sit'N Listen - In their podcast, the SITN team brings in scientists...The Goal of the SITN Podcast - Vini and Amy would like to develop the SITN podcast as a means for listeners...senior.  To learn more about the STIN's podcast, email sitnpodcast@gmail.com    For more info about starting...have to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...of Science in the News, and member of the SITN podcast team. You can follow Vini on twitter @vinidhra....Science in the News, and is a member of the SITN podcast team. You can follow Amy on twitter @aigilson. ...the audio engineer and producer for the Addgene Podcast.     Additional Resources on the Addgene Blog ...
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