High-efficiency CRISPR genome-editing tools for multiplex editing
Ready-to-transfect GeneArt™ CRISPR Nuclease mRNA circumvents time-consuming cloning steps needed when using CRISPR vector systems. Simply co-transfect Cas9 mRNA with in vitro transcribed guide RNA (IVT gRNA) or a synthetic gRNA expression cassette. 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 by adding multiple gRNAs. The system is versatile and simple to use, and changing target specificity only requires a change in the gRNA design.
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Figure 1. Complete RNA workflow for CRISPR/Cas9-mediated genome editing using the GeneArt CRISPR Nuclease mRNA and in vitro transcribed gRNA.
How to obtain a gRNA sequence:
Genome editing with CRISPR technology requires a noncoding guide RNA (gRNA) in order to cleave genomic DNA at a target sequence of interest.
Use our GeneArt CRISPR Search and Design Tool to search our database of >600,000 gRNA sequences specific to every gene in the human and mouse genomes. GeneArt predesigned gRNAs are optimized for gene knockout. Search results include recommendations based on minimizing potential off-target effects, potential binding sites, and exon maps with gRNA locations. Use this tool to analyze any sequence of interest and design unique CRISPR sequences.
Choose one of three options for making your gRNA:
1. The GeneArt Precision gRNA Synthesis Kit for transfection-ready gRNA in as little as four hours including template assembly.
2. GeneArt CRISPR Strings, a synthetic DNA template consisting of the gRNA with a T7 promoter for in vitro transcription or U6 promoter for ready-to-transfect format, which may be purchased via the GeneArt CRISPR Search & Design Tool or by completing the GeneArt CRISPR Strings DNA Order Form and submitting it to firstname.lastname@example.org.
3. Save time and effort and have our GeneArt custom services team design, synthesize, and purify in vitro transcribed (IVT) gRNA sequences for you. To obtain a services quotation, or to order, please contact our Custom Services department at 1-800-955-6288 x45682 or email@example.com.
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 2. 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 3. 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 4. 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 5. 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.
Figure 6. 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).
Need assistance with CRISPR gRNA design? Let our GeneArt CRISPR Search and Design Tool guide you to optimal designs. Start designing today
|GeneArt Strings U6 DNA
||> 200 ng
|GeneArt Strings T7 DNA
||> 200 ng
|Custom in vitro transcribed gRNA
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