High-efficiency CRISPR genome-editing tools for multiplex editing
The GeneArt® CRISPR Nuclease mRNA is wild type Cas9 as mRNA, for genome editing using CRISPR-Cas9 technology. Ready-to-transfect Cas9 mRNA circumvents the need for time-consuming cloning steps required when using CRISPR vector systems. Cas9 mRNA can be co-transfected with in vitro transcribed guide RNA (IVT gRNA) or a synthetic gRNA expression cassette containing U6 promoter. The gRNA expressing cassette and IVT gRNA template can be ordered as GeneArt® CRISPR Strings™ DNA, a 500 bp DNA fragment. Following transfection, the Cas9 protein is directed by gRNA to target specific genomic locus. This system allows multiplex genome editing, where multiple target gene sequences can be edited simultaneously in a single transfection reaction with the addition of multiple gRNAs. The system is versatile and simple to use, and changing target specificity only requires a change in the design of the GeneArt® CRISPR Strings™ DNA.
CRISPR Strings™ DNA–ready-to-transfect format or complete RNA system
GeneArt® CRISPR Strings™ DNA fragments are custom-designed to generate the CRISPR guide RNA (gRNA) component of the CRISPR-Cas9 system. They are offered with either a U6 or T7 promoter. The GeneArt® CRISPR U6 Strings™ DNA can be directly introduced into the cells (in case of GeneArt® CRISPR U6 Strings™ DNA) along with Cas9 mRNA, used as a template for generating in vitro transcribed gRNA (GeneArt® CRISPR T7 Strings™ DNA) or can be cloned into a vector to generate a gRNA expression plasmid. The U6 promoter drives expression of CRISPR gRNA for complexing with the Cas9 protein generated from the Cas9 mRNA. GeneArt® CRISPR T7 Strings™ DNA can be in vitro transcribed into gRNA using our MEGAshortscript™ T7 Transcription Kit prior to transfection. The gRNA is then combined with the Cas9 mRNA for a complete RNA format for more difficult-to-transfect cells and elimination of promoter constraints, for genome editing in a broader range of cell types. For broad cell type applications and high genome editing efficiency, we recommend using the complete RNA format (GeneArt® CRISPR nuclease mRNA and in vitro transcribed gRNA) along with Lipofectamine® MessengerMax reagent.
GeneArt® CRISPR Strings™ DNA must be purchased separately. We can help design your optimal target sequence for you—simply download the GeneArt® CRISPR Strings™ DNA order form and submit it with any design questions to firstname.lastname@example.org. Aditionally, we can design and in vitro transcribe your gRNAs, providing you with ready-to-transfect gRNAs and CRISPR mRNA.
|Figure 1. Workflow for ready-to-transfect format. Workflow for GeneArt® CRISPR mRNA & GeneArt® U6 Strings™ DNA. Three GeneArt® U6 Strings™ DNA fragments are co-transfected with Cas9 mRNA in the example here, allowing for multiple gene edits within one reaction. The CRISPR Strings™ DNA (500 bp synthetic DNA expression cassette) containing a U6 promoter can be directly transfected into the cell with Cas9 mRNA. The U6 promoter drives expression of gRNA that forms a complex with the Cas9 protein generated from the Cas9 mRNA. The use of the human Pol III promoter U6 ensures high expression of noncoding gRNA. The method of transfection varies based on cell type. For high editing efficiency, use Lipofectamine® 3000 transfection reagent.|
|Figure 2. Workflow for complete RNA format. Workflow for GeneArt® CRISPR mRNA & GeneArt® T7 Strings™ DNA. Three different in vitro transcribed (IVT) gRNAs are co-transfected with Cas9 mRNA in the example here, allowing for multiple gene edits within one reaction. CRISPR Strings™ fragments with a T7 promoter (500 bp synthetic DNA expression cassette) can be in vitro transcribed into gRNA using our MEGAshortscript™ T7 Transcription Kit prior to transfection. IVT gRNA together with Cas9 mRNA provides a complete RNA system that eliminates promoter constraints and allows high genome-editing efficiencies across a broader range of cell types. This system is suitable for in vivo applications such as microinjection and developing model systems. The method of transfection varies based on cell type. For high editing efficiency, use the complete RNA format with Lipofectamine® MessengerMax reagent, an RNA-specific transfection reagent for a broad range of cell types.|
How to order
Order U6 ready-to-transfect format
Each CRISPR Strings™ DNA will be designed against your target sequence and returned for your review.
Alternatively, known CRISPR sequences can be entered for synthesis into a CRISPR Strings™ DNA backbone.
Monitoring CRISPR genome-editing success
CRISPR-Cas9–mediated cleavage efficiency in a broad range of cell types
Cleavage efficiency can be detected using the GeneArt® Genomic Cleavage Detection Kit, which uses an assay that leverages mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.
Figure 3. The GeneArt® CRISPR Nuclease mRNA system demonstrates efficient genome editing in a broad range of cell types. Shown here are results from three different human cell lines—(A) 293FT; (B) HCT116; (C) U2OS—that were transfected in 24-well format to target the HPRT or RelA locus. CRISPR formats used and corresponding sample lane numbers are listed in the respective tables. The GeneArt® CRISPR Nuclease OFP vector was transfected using Lipofectamine® 3000 reagent, whereas other formats were transfected using Lipofectamine® MessengerMax reagent. At 72 hours post-transfection, cells were harvested and genome-editing efficiency was quantified using the GeneArt® Genomic Cleavage Detection Kit. The cleaved lower molecular mass band is indicated in the gel image with an arrow.
Broad host and stem cell applications
The complete RNA format along with the Lipofectamine® MessengerMax transfection reagent provides superior delivery combined with high genome-editing efficiency in stem cells and difficult-to-transfect cell lines.
Figure 4. The GeneArt® CRISPR Nuclease mRNA system has broad-host genome-editing applications. Shown here are results for gene editing at the ROSA26 and NANOG locus in mouse Neuro 2A cells that were transfected in 24-well format using Lipofectamine® 2000 reagent and analyzed 72 hours posttransfection using the GeneArt® Genomic Cleavage Detection Kit.
Figure 5. The GeneArt® CRISPR Nuclease mRNA system combined with in vitro transcribed gRNA produces high genome-editing efficiency in iPSCs, compared to other CRISPR RNA formats tested. We recommend using Lipofectamine® MessengerMax reagent for superior transfection and cleavage efficiency. Cells were transfected in 24-well format and harvested 72 hours posttransfection. Genome editing efficiency was quantified using the GeneArt® Genomic Cleavage Detection Kit.
Efficient multiplexing system enables reduced hands-on time with a simple workflow
Simultaneously transfect up to 4 targets in a single well, and assess the cleavage efficiency of multiple genes simultaneously. The Cas9 mRNA can be used in multiplexing approaches with more than one GeneArt® CRISPR Strings™ DNA fragment. Use this approach to determine which gRNA sequence works best for a particular target, or edit multiple genomic loci with one transfection.
Figure 6. Multiplexed genome editing using GeneArt® CRISPR mRNA and GeneArt® U6 String DNA. Genome-editing efficiency in 293 cells transfected with GeneArt® CRISPR Nuclease reporter plasmid or cells treated with GeneArt® CRISPR Nuclease mRNA + U6 gRNA string at one target or simultaneously targeted with either double or triple targets at the HPRT, AAVS1, and/or EMX1 genes.
Other potential applications of Cas9 mRNA include generation of transgenic model systems
While we haven’t tested the use of GeneArt® CRISPR nuclease mRNA in microinjections or other in vivo–mediated delivery methods for transgenic model system generation, many citations show its use in in vivo applications in a wide variety of organisms, including mouse, zebrafish, and Drosophila. For microinjection experiments and mouse model generation we recommend testing at least 3 gRNAs and validating the gRNAs in cell line of choice. Using GeneArt® CRISPR Strings™ DNA allows you to simultaneously screen multiple gRNA in a single transfection. Following validation you can clone your validated gRNA into an expression plasmid and make IVT gRNA template from sequence verified plasmids. See user manual for detailed instructions on generating synthetic PCR templates from gRNA expression plasmids.
GeneArt® CRISPR mRNA
|GeneArt® CRISPR nuclease mRNA||15 μg||A25640|
|GeneArt® Strings™ U6 DNA||> 200 ng|
|GeneArt® Strings™ T7 DNA||> 200 ng|
|Custom in vitro transcribed gRNA|
|GeneArt® Genomic Cleavage Detection Kit||20 reactions||A24372|
|Lipofectamine® MessengerMax Reagent||0.3 mL||LMRNA003|
|Lipofectamine® 2000 Transfection Reagent||1.5 mL||11668019|
|Lipofectamine® 3000 Transfection Reagent||1.5 mL||L3000015|
|MEGAshortscript™ T7 Transcription Kit||25 reactions||AM1354|
|MEGAclear™ Transcription Clean-Up Kit||20 reactions||AM1908|
For Research Use Only. Not for use in diagnostic procedures.