Skip to main content
Addgene

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

In Category

We narrowed to 40 results for: cas9 single guide rna sgrna expression vector

Showing: 21 - 40 of 40 results

  1. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ...which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome editing in large-scale has led...multiplexed gRNA expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic...pAWp28 storage vector to the pAWp12 lentiviral vector should retain the diversity in your gRNA library, but..."Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology...screening in mammalian cells with a lentiviral CRISPR-guide RNA library." Nature biotechnology 32.3 (2014): 267...20-bp gRNA target sequence and its 8-bp barcode, respectively. Once cloned into the storage vector, the...barcoded gRNA library is now ready for CombiGEM-based assembly into the CombiGEM lentiviral vector backbone...

  2. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...assemble multiple unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This ...conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger expression cassettes ...chromosomal integration and gene expression, the system includes a toolkit to express recombinant proteins within...for the multiplexing of sgRNAs against unique genomic targets or multiple sgRNAs against the same target...the Rapid Design and Production of Diverse Expression Vectors.” ACS Synthetic Biology, vol. 6, no. 7, American...modifications which can be especially difficult in alternative expression systems such as bacteria. If your yeast...repurposed for CRISPR applications (gene insert = Cas9 + gRNA), Addgene distributes specialized CRISPR kits...

  3. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...constitutively express Cas9 with a library of sgRNA expression vectors (containing ten guides against each...contains Cas9, Cas9 nickase, and Cas9 activator expressing plasmids that can co-express 1-8 gRNAs. Ordon... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ... Liu lab’s own pAc-sgRNA-Cas9 insect expression backbone, which expresses the gRNA from a Drosophila U6...assembly to produce single plasmid targeting vectors that encode both a guide RNA transcript and a repair...protein expression constructs fused with epitopes or fluorophores, generate expression vectors with various...CRISPR-Cas9-based tool for targeting CpG methylation by fusing catalytically inactive Cas9 (dCas9) with...

  4. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was...reporters for single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9 efficiency...cells with the BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP conversion and...that expressed either Cas9 or an optimized cytidine base editor. The lab then introduced an sgRNA targeting...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...the APOBEC-Cas9 editosome would restore eGFP fluorescence, making it easy to quantify single base editing...enrichment (TREE) to purify single base edited cells without the need for single cell isolation and downstream...

  5. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16: . https://doi.org...Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background with Our CRISPR Guide Pages...have a single gene they want to activate, we would recommend using a cDNA overexpression vector rather...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...three or four guides directed towards that gene as there can be a large difference in guide potency. Thus...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 ...

  6. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector and a Cas9 expression plasmid...endonuclease Cas9 to create a DNA double stranded break (DSB) at a genomic site specified by a guide RNA (gRNA...then mixed and transfected along with a Cas9 expression vector. They can also be introduced directly into...cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag...cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, 2)...reaction to generate high-throughput targeting vectors. These vectors can be used to tag endogenous genes and...of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence for...

  7. CRISPR Plasmids - Drosophila

    Type
    Collection
    ...Promoter PI Publication Empty gRNA Expression Vectors Select a gRNA expression plasmid based on factors such...Epigenetics RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen...transcription Virmilion none, need Cas9 plasmid Bullock and Port 49330 pAc-sgRNA-Cas9 dU6 BspQI Transfection Puromycin...also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator...base-to-base conversions using a prime editing guide RNA (pegRNA). The pegRNA directs the nickase to the target site...same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their... to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas ...

  8. CRISPR References and Information

    Type
    Collection
    ... KB Zhang gRNA cloning CRISPR RNA array: Cas9 (pX260) or Cas9 D10A (pX334) ; tracrRNA: Cas9 (pX330) or...ssDNA oligos p201G Cas9 ; p201B Cas9 ; p201H Cas9 ; p201N Cas9 ; pUC gRNA Shuttle PDF, 500 KB Sabatini...sites for S. pyogenes Cas9, S. aureus Cas9, S. thermophilus Cas9, N. meningitidis Cas9, or Cas12a from your...Injection and selection for Cas9-triggered homologous recombination Cas9-sgRNA target construct ; pMA122...injection gRNA core ; Cas9 ; optimized Cas9 PDF, 66.8 KB Fujii gRNA design and cloning gRNA cloning vector...Nematode: Cas9 and gRNA use Cas9 (pIK86) ; gRNA empty backbone (pDR274) PDF, 89 KB Liu Fly: gRNA cloning...DNA FLAG tagged dCas9 PDF, 110 KB Goldstein Nematode: gRNA design and cloning Cas9-sgRNA construct PDF,...

  9. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    ...instead activates/represses gene expression by various mechanisms. The RNA guide is typically 20 nts and the...off-target effects? The CRISPR-Cas system is an RNA-guided nuclease that either introduces a ssDNA nick,...approaches have been used - GUIDE-Seq and integrase-defective lentiviral vectors - which rely on insertion...CRISPR Guide Addgene’s CRISPR 101 eBook More resources on the Addgene blog CRISPR 101: Which Cas9 do I...mutations can be mitigated by using two gRNAs in close proximity that guide Cas nickases. When two nicks are ...proximal to a PAM site for DNA cutting to occur. The RNA sequence and PAM site confer specificity of the nuclease...of 5%. Your experiment involves making a large sgRNA library to thousands of genes and targeting and ...

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

    Type
    Blog Post
    ...A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal... 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...too many Cas9 integrations per cell, and my stable cell pool had wildly variable Cas9 expression (Figure...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9,... 20% of their Cas9-expressing cells. So, even when Cas9 is efficiently editing, don’t expect a 100% knockout...

  11. CRISPR Guide

    Type
    Collection
    ...Science Guides CRISPR Guide CRISPR Guide Jump To: CRISPR Guide Overview CRISPR Basics Small Edits Large...components: a guide RNA ( gRNA or sgRNA ) and a CRISPR-associated endonuclease (Cas enzyme). The gRNA is a short...conversions using a prime editing guide RNA ( pegRNA ). Like sgRNA, pegRNA directs the nickase to the target...more about Cas9 multiplexing . Browse Plasmids: Multiplex gRNA Vectors Engineering the Cas9 Endonuclease...transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9 is included in the gRNA-containing...We’ll discuss more Cas9 fusion tools later in the guide. Increasing fidelity of Cas9 Researchers have also... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...

  12. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ... purifying Cas9? Check out pET-28b-Cas9-His from Alex Schier's lab, designed for expression and purification...compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional... is expressed along with Cas9. If the gRNA effectively cuts the target sequence, the plasmid undergoes... of Yonglong Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen and colleagues...colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain... existing transposon and vector systems and creating an all-synthetic vector that included only the elements...perform genome-wide knockout screens. These vectors and sgRNA libraries expand upon the CRISPR family of...

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

    Type
    Blog Post
    ...Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152:1173–1183...includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette....catalytically dead Cas9 (dCas9) alone or a dCas9 fused to the omega subunit of RNA polymerase, the lab...technology (Ronda et al., 2016). pMA7CR_2.0 expresses lambda Red and Cas9, which are separately inducible by L-arabinose...functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes in that Cas9-mediated cleavage induces ...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) and the Zhang lab's system...bacteria As RNA interference does not function in bacteria, most efforts to regulate gene expression were limited...

  14. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    ... 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target, the Cas9 endonuclease that... An alternative Cas9, from Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from...., 2016). AAV can also be used as a vector to deliver CRISPR/Cas9 either through injection into either...(2014) Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol... Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector. iScience 9:286–297...et al., 2018, Mizuno et al., 2018). Alternatively, the gRNA, Cas9, and donor oligonucleotide can be electroporated...online to guide you in gRNA design. We have been using Benchling, but there are many other gRNA design tools...

  15. Empty Backbones - Choosing Your Perfect Plasmid Backbone

    Type
    Collection
    ...compatible Y2H vector - BD pRPR1_gRNA_handle_RPR1t - gRNA expression empty vector pFA6a vectors - Epitope ...lentiviral vector for ORF expression pDest-565 - Gateway destination vector for bacterial expression; His,GST...astrocyte-selective expression in AAV Dual promoter Separate expression pRGEB31 - Deliver sgRNA and Cas9 into plants...gene expression vector URA3 Yeast pAG306GAL-ccdB - Yeast Gateway expression vector (see...Tet-inducible lentiviral shRNA expression pLKO.1 - TRC cloning vector - Lentiviral shRNA expression pLJM1-EGFP - ...hygromycin resistance pRGEB32 - Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter...transient expression vector GFP Varies pMKO.1 GFP - Retroviral shRNA expression pLenti CMV...

  16. CRISPR Plasmids - Tagging

    Type
    Collection
    ...PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh - expresses Cas9, PITCh-gRNA, & FBL-specific gRNA pCRIS-PITChv2...reaction produces a single plasmid targeting vector that encodes both a guide RNA transcript and a repair...Epigenetics RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen...targeting vector assembly, and the remaining 5 plasmids include Cre and FLP expression vectors, a general...uses PCR to generate both an expression cassette for the Cas9-programming sgRNA and HR donors for selectable...components: A vector containing Cas9 and a validated gRNA, based on the Zhang lab's PX458 . Multiple gRNA plasmids...you will need to design and clone in a gRNA(s) to guide the Cas9 protein to your target sequence, as well...

  17. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    ... modular toolkits build single plasmid targeting vectors that encode the gRNA transcript and the repair...co-conversion markers. Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins...their seminal work on micro RNAs in C. elegans. The C. elegans work on small RNAs informed and drove work ...and yeast cells using modified Cas9 proteins to regulate gene expression (CRISPRi/CRISPRa), visualize specific...Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis...aspects of editing. First, they devised guidelines for designing single-stranded repair templates (Farboud ...University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies have transformed the palette...

  18. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    ...It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like...Differentially expressed genes  As the costs for global gene expression analyses like RNAseq are getting ...test which of the differentially expressed genes found in a gene expression dataset are causal for a phenotype...differentially expressed genes that are known drug targets, or all differentially expressed genes that code..."Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9." Nature biotechnology...generation sequencing to identify which gRNAs are differentially expressed in the separate groups and, as a ...for this purpose.  The gRNA oligos are subsequently cloned into your vector of choice using the adapter...

  19. CRISPR Guide

    Type
    Guide
    ...Science Guides CRISPR Guide CRISPR Guide Jump To: CRISPR Guide Overview CRISPR Basics Small Edits Large...components: a guide RNA ( gRNA or sgRNA ) and a CRISPR-associated endonuclease (Cas enzyme). The gRNA is a short...conversions using a prime editing guide RNA ( pegRNA ). Like sgRNA, pegRNA directs the nickase to the target...more about Cas9 multiplexing . Browse Plasmids: Multiplex gRNA Vectors Engineering the Cas9 Endonuclease...transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9 is included in the gRNA-containing...We’ll discuss more Cas9 fusion tools later in the guide. Increasing fidelity of Cas9 Researchers have also... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...

  20. Lentiviral Vector Guide

    Type
    Guide
    ... Guides Lentiviral Vector Guide Lentiviral Vector Guide Jump To: Lentiviral Vectors Lentiviral...gamma-retroviruses, see Addgene's gamma-retroviral vector guide . Lentiviral Vectors The genome of lentiviruses typically...Lentiviral Vectors Due to their integration and long-term expression of a transgene, lentiviral vectors are ...Viral Vectors 101 topics More Lentiviral Vector topics Addgene Protocols pLKO.1 - TRC Cloning Vector Protocols...gene silencing by RNAi in primary cells. RNA , 9 (4), 493–501. https://doi.org/10.1261/rna.2192803 PMID: ...lentiviral vectors that can be safely used in laboratory settings. These engineered lentiviral vectors have ...models. Lentiviral vectors can target non-dividing cells, enable stable long-term expression in a host, and...
Showing: 21 - 40 of 40 results