GeneArt® CRISPR Nuclease mRNA

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 transfected into cells with Cas9 mRNA. 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 geneartsupport@lifetech.com. Aditionally, we can design and in vitro transcribe your gRNAs, providing you with ready-to-transfect gRNAs and CRISPR mRNA.

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.

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.

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.

Figure 7. Highly efficient multiplexed genome editing using Cas9 mRNA and in vitro transcribed gRNA. Shown here are the editing efficiencies from either cells transfected with an all-in-one plasmid or cells treated with just one target or simultaneously targeted with either double or triple targets. We also observe RelA-specific cleavage in all the samples co-transfected with RelA in either single or multiplex format (results not included).

For Research Use Only. Not for use in diagnostic procedures.