Multiplex gene editing with CRISPR Nuclease mRNA


GeneArt CRISPR Nuclease mRNA is a ready-to-transfect wild-type Cas9 mRNA for performing CRISPR/Cas9-mediated genome editing. When co-transfected with a guide RNA (gRNA) that targets your genomic locus of choice, the Cas9 protein is directed by the gRNA to cleave the gene at that locus.

The CRISPR Nuclease mRNA system allows multiplex genome editing, in which 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. Changing the target requires only a change in the gRNA design.
 

GeneArt CRISPR Nuclease mRNA

TIP: If you’re new to CRISPR editing, we recommend using TrueCut Cas9 Proteins as your nuclease. The protein process does not require mRNA translation and allows you to deliver a ready-to-edit Cas9 ribonucleoprotein complex.

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NOTE: CRISPR-Cas9 genome editing requires a guide RNA (gRNA) in order to cleave genomic DNA at a target sequence of interest. Consult Guide RNAs for CRISPR-Cas9 and CRISPR Cas9 Screening Libraries for more information.  

Cas9 mRNA workflow for multiplex gene editing and transgenic model system applications

With GeneArt CRISPR Nuclease mRNA, you can simultaneously transfect up to four different gRNAs in a single well and assess the cleavage efficiency of multiple genes simultaneously. You can use this approach to determine which gRNA sequence works best for a particular target, or to edit multiple genomic loci with one transfection.

In addition, CRISPR-Cas9 genome editing using Cas9 mRNA has been cited for use with microinjections and other in vivo-mediated delivery methods for transgenic model system generation in a wide variety of organisms, including mice [1], zebrafish [2], and Drosophila. For microinjection experiments and mouse model generation, we recommend testing at least three synthetic sgRNAs and validating them in the cell line of choice.
 

Flow chart illustrating the use of CRISPR mRNA for multiplexed gene editing along with a bar graph and gel image showing that cleavage efficiency for each target gene is consistent when using GeneArt CRISPR Nuclease mRNA and IVT gRNA to target only a single gene or multiple genes
Flow chart illustrating the use of CRISPR mRNA for multiplexed gene editing along with a bar graph and gel image showing that cleavage efficiency for each target gene is consistent when using GeneArt CRISPR Nuclease mRNA and IVT gRNA to target only a single gene or multiple genes

Figure 1. Efficient multiplexed genome editing using GeneArt CRISPR Nuclease mRNA and IVT gRNA. Genome editing efficiency is shown in HEK293 (human embryonic kidney) cells transfected with GeneArt CRISPR Nuclease mRNA and in vitro transcribed (IVT) gRNA at one target (red) or simultaneously targeted with either double (yellow) or triple (green) targets at the HPRT, AAVS1, and/or RelA genes. Control cells transfected with IVT gRNA only (gray) and cells transfected with GeneArt CRISPR Nuclease reporter plasmid (purple) are shown for comparison. Editing efficiency was consistent across conditions, regardless of the number of targets.
 

CRISPR mRNA applications across hosts and in transgenic model systems

Data: GeneArt CRISPR Nuclease mRNA achieves high editing efficiency in mouse neural cells

The complete Cas9 mRNA format, delivered with our Lipofectamine transfection reagents, provides superior delivery combined with high genome editing efficiency in stem cells and difficult-to-transfect cell lines.

Bar graph and gel image showing higher cleavage efficiency in cells transfected with Cas9 mRNA and IVT gRNA than cells transfected with the all-in-one CRISPR plasmid

Figure 2. The GeneArt CRISPR Nuclease mRNA system enables efficient genome editing across hosts. The system was used with IVT gRNA to edit Mouse Neuro-2A (mouse neuroblast) cells at the ROSA26 and NANOG loci. Cells were transfected in 24-well format using Lipofectamine 2000 Reagent and analyzed 72 hours post-transfection using the GeneArt Genomic Cleavage Detection Kit. For both genes, cleavage efficiency exceeded the all-in-one CRISPR plasmid.
 

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References
  1. Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013; 13: 659–662. 
  2. Shah AN, Davey CF, Whitebirch AC, Miller AC, Moens CB. Rapid reverse genetic screening using CRISPR in zebrafish. Nat Methods. 2015; 12: 535­–540. 
  3. Hashimoto M, Takemoto T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep. 2015; 5: 11315. 
     
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