Get up to 10X higher cleavage efficiency using CRISPR mRNAs in stem cells

Genome editing in induced pluripotent stem cells (iPSCs) has been demonstrated to be highly effective for generating disease models for both monogenic and complex genetic disorders. For successful genome editing and downstream application of iPSCs, many factors need to be considered, such as choice of growth media, extracellular matrix, genome editing tools, and nucleic acid (NA) delivery methods (Figure 1).

Lipofectamine™ MessengerMAX™ reagent increases the likelihood of cleavage and recombination with GeneArt™ CRISPR Nuclease mRNA through highly efficient transfection, maximizing the efficiency of CRISPR-driven genetic modifications and simplifying downstream processes (see figure 2).

Figure 1. Factors affecting genome editing outcomes in iPSCs. Here we show feeder-free culture of stem cells and a genome editing protocol that can facilitate efficient disease model generation. Download our PDF on robust genome editing in stem cells using GeneArt CRISPR Nuclease mRNA.

Figure 2. Cleavage efficiency of various GeneArt™ CRISPR formats targeting the HPRT locus in Gibco™ iPS cells in a 12-well format. Lipofectamine™ 3000 reagent was used to deliver CRISPR nuclease all-in-one plasmid DNA; Lipofectamine™ MessengerMAX™ and two leading mRNA delivery reagents were used to deliver an all-RNA CRISPR format (Cas9 mRNA + IVT gRNA). Cleavage efficiency was determined using the GeneArt® Genomic Cleavage Detection Kit 72 hours post-transfection.

Watch the video to discover how to intgrate Lipofectamine MessengerMAX into your workflow

"Lipofectamine® MessengerMAX™ reagent can transfect mesenchymal stem cells at high efficiency wiithout the toxic effects of viral vectors." Dr. Daniel Cohen, University of Pennsylvania

"I tested the MessengerMAX on rat pancreatic primary alpha and beta cells, using a positive control eGFP. The expression worked perfectly. I then checked my target gene expression and it increased by more than 1000 fold!" (see data) Dr Mounia Heddad-Masson, Hospital of Geneva (diabetology dpt.)

CRISPR-Cas–Based Genome Editing: Technology Overview

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems are revolutionizing the field of genome editing. Able to achieve highly flexible and specific targeting, CRISPR-Cas systems can be modified and redirected to become powerful tools for genome editing in many species, including mammalian cells. It is a simple system comprising a catalytic unit, Cas9, and a short noncoding guide RNA (gRNA) that confers target specificity. It is an attractive tool for large-scale genome engineering in a wide variety of hosts. We have developed various CRISPR-Cas9 formats that can be used to edit genomes in a wide variety of cell types, including stem cells. Using these formats we have achieved greater than 50% target-specific DNA cleavage in mouse embryonic stem cells (ESCs) and human iPSCs and ESCs. With so many genomic tools and technologies available, trying to decide which ones to use for your research can be overwhelming. That’s why we’ve simplified the process with this easy to use selection guide.

Amazing transfection efficiency in neurons and primary cell types

Lipofectamine® MessengerMAX™ mRNA Transfection Reagent delivers amazing transfection efficiency in neurons and a broad-spectrum of primary cells, enabling improved application outcomes and more biologically relevant research. That's because our novel lipid nanoparticle technology is optimized to deliver the highest amount of mRNA possible without the nuclear entry step that is required with DNA.

Figure 3. Lipofectamine® MessengerMAX™ outperforms leading DNA delivery reagent and leading mRNA delivery reagent in primary cells (fresh isolated mouse cortical neurons, BJ fibroblasts, and primary keratinocytes). Lipofectamine® MessengerMAX™ and the leading mRNA delivery reagent were used to deliver GFP mRNA (500 ng/well) in a 24-well format. The leading DNA delivery reagent was used to deliver GFP DNA (500 ng/well), and GFP was analyzed 24-hours posttransfection.

Figure 4. Lipofectamine® MessengerMAX™ outperforms leading DNA delivery reagent and leading mRNA delivery reagent in various stem cells (Gibco® iPSCs, H9 ESCs, mESCs, hNSCs). Lipofectamine® MessengerMAX™ and the leading mRNA delivery reagent were used to deliver GFP mRNA (250 ng/well) in a 48-well format. The leading DNA delivery reagent was used to deliver GFP DNA (250 ng/well), and GFP was analyzed 24-hours posttransfection.

Figure 5. Lipofectamine® MessengerMAX™ outperforms leading DNA delivery reagent and leading mRNA delivery reagent in various cell lines that are very difficult to transfect (bEnd.3, SH-SY5Y, SK-N-SH, A431, RAW 264.7 and HT29). Lipofectamine® MessengerMAX™ and the leading mRNA delivery reagent were used to deliver GFP mRNA (500 ng/well) in a 24-well format. The leading DNA delivery reagent was used to deliver GFP DNA (500 ng/well), and GFP was analyzed 24-hours posttransfection.

Figure 3. Lipofectamine® MessengerMAX™ outperforms leading DNA delivery reagent and leading mRNA delivery reagent in primary cells (fresh isolated mouse cortical neurons, BJ fibroblasts, and primary keratinocytes). Lipofectamine® MessengerMAX™ and the leading mRNA delivery reagent were used to deliver GFP mRNA (500 ng/well) in a 24-well format. The leading DNA delivery reagent was used to deliver GFP DNA (500 ng/well), and GFP was analyzed 24-hours posttransfection.

Figure 4. Lipofectamine® MessengerMAX™ outperforms leading DNA delivery reagent and leading mRNA delivery reagent in various stem cells (Gibco® iPSCs, H9 ESCs, mESCs, hNSCs). Lipofectamine® MessengerMAX™ and the leading mRNA delivery reagent were used to deliver GFP mRNA (250 ng/well) in a 48-well format. The leading DNA delivery reagent was used to deliver GFP DNA (250 ng/well), and GFP was analyzed 24-hours posttransfection.

Figure 5. Lipofectamine® MessengerMAX™ outperforms leading DNA delivery reagent and leading mRNA delivery reagent in various cell lines that are very difficult to transfect (bEnd.3, SH-SY5Y, SK-N-SH, A431, RAW 264.7 and HT29). Lipofectamine® MessengerMAX™ and the leading mRNA delivery reagent were used to deliver GFP mRNA (500 ng/well) in a 24-well format. The leading DNA delivery reagent was used to deliver GFP DNA (500 ng/well), and GFP was analyzed 24-hours posttransfection.