CRISPR-Cas9 Gene Editing
Optimized tools for high-confidence genome engineering
The Dharmacon Edit-R CRISPR-Cas9 platform greatly simplifies the workflow of permanently knocking out genes. Our approach includes predesigned, ready-to-use DNA and RNA components and enables fast assessment of multiple target sites per gene for multiple genes.
Explore our products for successful CRISPR-Cas9 genome engineering
High quality, ready-to-use lentiviral and synthetic reagents to guide Cas9 cleavage
Configure the optimal promoter for your cell type to ensure robust Cas9 expression or explore DNA-free options
Proper controls are essential to assessment of CRISPR-Cas9 genomic editing experiments
Pooled sgRNA or arrayed crRNA for high-throughput gene editing studies
Synthetic single-stranded DNA oligonucleotide (ssDNA oligo) donors for precise genomic modification with the homology-directed repair (HDR) pathway
Introduction to CRISPR
The ability to precisely and permanently alter endogenous gene expression through targeted genome editing is a highly effective reverse genetics tool. Recently, genome engineering has advanced tremendously with the characterization of bacterial and archael CRISPR (clustered regularly interspaced short palindromic repeats) systems and their adapted usage in mammalian cells. (Figure 1)
Type II CRISPR-Cas9 system
Figure 1. A Type II CRISPR-Cas9 system generally consists of the Cas9 nuclease complex programmed by tracrRNA and crRNA. As such, it is easily adapted for genome engineering in mammalian cells.
CRISPR systems have been described in the literature as innate immune defense systems analagous to eukaryotic RNA interference (RNAi) pathways. Additionally, the Cas9 (CRISPR-associated 9) nuclease has been defined as a dedicated effector enzyme that cleaves DNA when guided by two required small RNA sequences: the CRISPR RNA (crRNA) which binds the target DNA and guides cleavage, and the trans-activating RNA (tracrRNA) which base-pairs with the crRNA and enables the Cas9-crRNA complex to locate the targeted DNA. Recent publications demonstrate this system can be engineered to target and cleave DNA in mammalian cells, thereby permanently disrupting gene expression, making this system a new and exciting molecular tool to interrogate gene function.
Below is a selection of important journal articles in the CRISPR-Cas9 research field.
- R. Barrangou, A. Birmingham et. al. Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference. Nucleic Acids Res. 43(7), 3407-3419 (2015)
- D. Bhaya, M. Davison, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45, 273-297 (2011).
- L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339(6121), 819-823 (2013).
- E. Deltcheva, K. Chylinski, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 471(7340), 602-607 (2011).
- Y. Fu, J. D. Sander, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. (2014).
- D.Y. Guschin, A. J. Waite, et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247-256 (2010).
- F. Heigwer, G. Kerr, et al. E-CRISP: fast CRISPR target site identification. Nat. Methods. 11(2), 122-123 (2014).
- P.D. Hsu, D. A. Scott, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31(9), 827-832 (2013).
- M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337(6096), 816-821 (2012).
- P. Mali, L. Yang, et al. RNA-guided human genome engineering via Cas9. Science. 339(6121), 823-826 (2013).
- N. K. Pyzocha, F. A. Ran, et al. RNA-Guided Genome Editing of Mammalian Cells. Methods Mol. Biol. 1114, 269-277 (2014).
- D. Reyon, C. Khayter, et al. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly. Curr. Protoc. Mol. Biol. 100, 12.15.1‐12.15.14 (2012).
- T. R. Sampson, D. S. Weiss. Exploiting CRISPR/Cas systems for biotechnology. Bioessays. 36(1), 34-38 (2014).
- T. Wang, J. J. Wei, et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 343(6166), 80-84 (2014).
CRISPR-Cas9 Gene Editing Products
Synthetic Guide RNA
Algorithm-optimized crRNA for genome-wide coverage of human, mouse, or rat genes. Simply search for your gene!
The Edit-R trans-activating CRISPR RNA (tracrRNA) is a chemically synthesized and HPLC-purified long RNA required for use with synthetic crRNA to form the complex that programs Cas9 nuclease.
Species-specific crRNAs targeting well-characterized genes, as well as mismatch detection assay primers, to determine the effectiveness of your gene editing conditions for maximal efficiency.
Non-targeting controls to evaluate cellular responses to CRISPR-Cas9 components in the absence of gene target-specific crRNA.
Plated pre-defined collections of popular human and mouse gene families for arrayed knockout screening.
Customize and order plates of predesigned crRNA for knockout studies for your targets of interest.
Lentiviral Guide RNA
Algorithm-optimized sgRNA for genome-wide coverage of human, mouse, or rat
genes. Provided as high-titer lentiviral particles and glycerol stocks.
Species-specific sgRNAs targeting well-characterized genes to determine the effectiveness of your gene editing conditions for maximal efficiency.
Non-targeting controls to evaluate cellular responses to CRISPR-Cas9 components in the absence of gene target-specific sgRNA.
High-titer pooled screening libraries for pre-defined gene sets in human and mouse.
Arrayed collections of lentiviral sgRNA libraries for high-throughput knockout screening across entire human gene families.
Vector-based Cas9 Nuclease
Non-lentiviral vectors provided as endotoxin-free purified DNA for direct co-transfection with Edit-R synthetic crRNA and tracrRNA.
Purified lentiviral particles or plasmid DNA for generation of stable Cas9 nuclease-expressing cell populations. Constitutive or inducible promoter options are available.
DNA-free Cas9 Nuclease
Custom Guide RNA Ordering & Design Tool
Place a custom order, or design and order your own crRNA or lentiviral sgRNA with our easy-to-use interface.
Use this tool to design and order a single-stranded DNA donor (≤ 150 nt) for precise CRISPR-Cas9 gene editing with the HDR pathway.
The breadth of the Dharmacon portfolio supports multiple gene editing workflows; choose the one that suits your experimental system and desired outcome.
Gene knockout workflow using the Edit-R Cas9 Nuclease mRNA with synthetic crRNA and tracrRNA
Gene editing with Edit-R Cas9 Nuclease mRNA or protein and crRNA:tracrRNA is performed by co-transfecting all components with DharmaFECT Duo Transfection Reagent (or other DharmaFECT transfection reagent suitable for your specific cells of interest). One may then observe phenotypes directly. A DNA mismatch detection assay can be used to estimate gene editing efficiency prior to clonal cell line generation and characterization.
Gene knockout workflow using Cas9 Expression plasmid and Edit-R crRNA:tracrRNA
Gene editing with Edit-R Cas9 Nuclease Expression Plasmid and crRNA:tracrRNA is performed by co-transfecting all components with DharmaFECT Duo Transfection Reagent. One may then observe phenotypes directly (no enrichment), or enrich for transfected cells, either with cell sorting (with mKate2 plasmid) or puromycin selection (with PuroR plasmid). A DNA mismatch detection assay can be used to estimate gene editing efficiency prior to clonal cell line generation and characterization.
Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease Expression particles with synthetic crRNA and tracrRNA
Gene editing with Edit-R Lentiviral Cas9 Nuclease and crRNA:tracrRNA can be carried out in two ways. The left side of the diagram shows enrichment and expansion of the population of cells transduced with Edit-R Lentiviral Cas9 nuclease, then transfected with crRNA:tracrRNA for target gene editing. The right side of the diagram illustrates clonal isolation and expansion to create a more defined Cas9-expressing assay system. Both methods enable straightforward assessment of multiple target sites for multiple genes.
Gene knockout workflow using the Edit-R Lentiviral Cas9 Nuclease with Lentiviral sgRNA
Gene editing with Edit-R Lentiviral Cas9 Nuclease and sgRNA can be done following a mixed cell populations approach (left side), typically for gene knockout screening, or on isolated clonal cells lines (right side) when preferred or required.
CRISPR-Cas9 Gene Editing Resources
CRISPR RNA Configurator
Do you have a favorite guide RNA sequence?
Do you require design assistance to ensure Cas9 cleavage in a specific region of your target gene?
Edit-R predesigned crRNA and lentiviral sgRNA provide algorithm-optimized designs for functionality and specificity; but when particular designs or parameters are desired, you can design your own crRNA or sgRNA with our easy-to-use interface.
The Dharmacon CRISPR RNA Configurator allows you to design or enter your own CRISPR targeting sequences for incorporation into synthetic crRNAs and single guide RNAs (sgRNAs).
See the CRISPR RNA Configurator User Guide for detailed instructions.