Genome Editing Technology
Over the past several years, the advances in genome editing technologies such as Zinc Finger Nucleases (ZFN), Transcription Activator-like Effector Nuclease (TALENs) and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 Nuclease have vastly expanded a researcher’s toolkit for exploring gene function and shown potential for therapeutics.
One thing all the technologies have in common is the need to get the functional parts of the tools delivered into the cells in which the editing will take place. The optimal tool for facilitating this delivery can vary dependent on what is to be delivered, i.e. DNA, RNA, protein, size of the substrate, and to what cell type it is to be delivered. The goal is to deliver effectively such that a high percentage of the cells treated effect the edit and yet remain viable for downstream screening and analysis in the case of research or potentially to be returned to a patient in the case of therapeutics. The delivery method itself should have little to no effect on the viability and functionality of the cell. Of course, an optimal method for delivery is not as simple as the reagent or equipment used but can include culture conditions prior to or after for optimal delivery, recovery, and selection, as needed.
Use the following links to jump down to the relevant section:
- Genome Editing in Pluripotent Stem Cells
- Clonal Expansion of Pluripotent Stem Cells Lines Starting from a Single Cell
- Genome Editing in Neural Stem Cells
- Genome Editing in Adherent Primary Cells and Cell Lines
Pluripotent stem cells have come to the forefront of research as a cell type useful for study of cell differentiation or as a source for human differentiated cell types that are difficult to access to study gene function and as a potential source for cell therapy. Thus, there is high interest in performing genome editing in pluripotent stem cells. Transfection of pluripotent stem cells has been notoriously difficult and the special culture requirements for maintaining pluripotency present challenges for clonal isolation, expansion and analysis of the products of gene editing.
At MTI-GlobalStem we have optimized reagents for delivering gene editing tools that are DNA, mRNA and /or protein into stem cells to give you flexibility based on your particular genome editing method of choice. For pluripotent stem cells we offer a complete system for delivery, recovery , and clonal isolation in order to maximize gene editing in pluripotent stem cells.
PluriQ™ G9 Gene Editing System (GSK-9003), a complete system for culturing and transfecting human pluripotent stem cells for gene editing. Includes the following components:
- EditPro™ Stem Transfection Reagent, optimized for the delivery of DNA, RNA and/or protein into stem cells and tested specifically for delivery of CRISPR/Cas9 editing via DNA plasmid, mRNA Cas 9 with tracr/guide RNA, or Cas 9 protein with tracr/guide RNA. It is effective for TALEN and ZFN editing as well.
- PluriQ™ G9™ Maintenance Medium (GSK-9001), a defined, xeno-free, feeder-free medium used for expansion and maintenance of human ES and human iPS cells in vitro. Includes PluriQ™ G9™ Basal Medium (500 ml), PluriQ™ G9™ Supplement (50X), Human FGF-2, Human TGF-β1.
- G9™ Vitronectin (VTN), Human Recombinant, a substrate for plate coating. Validated to facilitate evenly spread cells across a monolayer to enhance efficiency and evenness of transfection.
- G9™ Versene Solution (1x), for passaging of stem cells, allows for easy, non-enzymatic, gentle passaging of the cells and is compatible with scale-up to higher throughput cell culture.
EditPro™ Stem Transfection Reagent (GST-2174) optimized for the delivery of DNA, RNA and/or protein into pluripotent and neural stem cells
PluriQ™ G9™ Cloning Medium (100ml) supports single cell/well clonal selection for human iPS or human ES cells.
CRISPR/Cas9 DNA Delivery
In Figure 1 below, we demonstrate the delivery of a typical CRISPR/Cas9 plasmid containing a GFP reporter by immunostaining for both the GFP expression and the Cas9 expression. This data was generated by transfecting iPSC cells in adherence grown on vitronectin in PluriQ™ G9™ Maintenance Medium such that cells are in a monolayer facilitating even, high efficiency transfection using EditPro™ Stem transfection reagent.
Fig. 1 CRISPR/Cas9 DNA Delivery with the PluriQ™ G9 Gene Editing System: Transfection with a standard CRISPR/Cas9 EF1a-GFP DNA construct. NCRM-iPS cells transfected using EditPro™ Stem with a ~10.5 kb plasmid expressing Cas9 and GFP.
Gene Editing by Co-Transfection using Cas9 modified mRNA or Cas9 RNP
In Figure 2, we again transfect adherent iPS cells grown on vitronectin in PluriQ™ G9™ Maintenance Medium. We use modified Cas9 mRNA or Cas9 Protein as our editing tools and include a modified GFP mRNA to track transfection efficiency. Cells were then assayed for successful editing via a T7Endo 1 Assay to detect small insertions and deletions (INDELs) . T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA. By amplifying the region of the edit in isolated genomic DNA followed by a final denaturation and annealing, one is able to detect the percentage of editing by the level of mismatch cleavage by the T7 Endonuclease. (Guschin, D.Y., et. al.(2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 649, 247–256.)
Fig. 2 PluriQ™ G9 Gene Editing System mediates INDEL formation in iPSCs: EditPro™ Stem Reagent was used to transfect cells with Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay.
Gene Editing by Co-Transfection using Cas9 modified mRNA or Cas9 RNP in Suspension (Reverse Transfection)
To allow flexibility in choice of pluripotent stem cell culture system, such as plating on other matrices or in other commercial media, we optimized transfection in suspension using a "reverse transfection" technique, for maximum transfection of all the cells in culture. Adherent cells grown in colonies on these matrices do not evenly transfect due to limited access of the reagent to the inner cells of the colony. Figure 3 illustrates how co-transfection of modified GFP mRNA and Cas9 RNP using the EditPro Stem reagent consistently produces >90% transfection efficiency as monitored by GFP in stem cells plated on vitronectin in PluriQ™ G9™ Maintenance Medium and on Geltrex in mTeSR™1.
Fig. 3 Transfection in Suspension: Expression of eGFP mRNA co-delivered with Cas9 Protein, tracrRNA and crRNAs Emx1 using EditPro™ Stem Reagent in human ESCs plated on A. vitronectin in PluriQ™ G9™ Maintenance Medium or B. Geltrex™ in mTeSR™
Using the transfection in suspension protocol, the modified Cas9 mRNA, tracrRNA and a modified GFP mRNA were co-transfected. The cells were then analyzed by T7Endo1 assay to look at the effectiveness of editing.
Fig. 4 Cas9 mRNA Transfection into iPSC Cells with EditPro™ Stem: 200,000 cells were transfected in suspension using EditPro™ Stem with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay.
Fig. 5 Cas9 Protein (RNP) Transfection of iPS or huES cells with EditPro™ Stem: 200,000 cells were transfected in suspension using EditPro™ Stem with Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay.
After genome editing, it is important to be able to isolate a clonal population containing the edit. PluriQ™ G9™ Cloning Medium supports single cell/well clonal selection for human iPS or human ES cells. Using G9™ Versene Solution (1x) for dissociation to single cells and dilution to 1-2 cells/well, it is possible to expand from single cells in PluriQ™ G9™ Cloning Medium and maintain pluripotency.
Fig. 6 PluriQ™ G9™ Cloning Medium. NCRM-1 subclone 485 after transfection with EditPro™ Stem, expanded from a single cell in G9 Cloning Medium on Vitronectin to a T75 flask in 4 passages, maintaining Oct4 expression a marker of pluripotency.
EditPro™ Stem provides superior delivery into neural stem cells. Using iPS-derived human neural stem cells and the protocol for transfection in suspension with subsequent plating on Geltrex in NeuralX™ NSC Medium Supplemented with GS22™ we see >90% efficiency by GFP reporter and excellent editing using either Cas9 mRNA or Cas9 RNP as evaluated by T7Endo 1 assay.
Fig. 7 Gene Editing by EditPro™ Stem Transfection of iPS-derived Neural Stem Cells: Neural Stem cells were transfected in suspension using EditPro™ Stem with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). GFP was observed after 24 hours. Genome-modification was analyzed using the T7Endo 1 assay.
DNA-In® CRISPR Transfection Reagent is optimized for large plasmid delivery and can be used to deliver CRISPR/Cas9 vectors into a range of cell types. This reagent is especially well suited for hard-to-transfect primary cells.
Fig. 8 DNA-In® CRISPR delivers Cas9-GFP expression vector with maximum efficiency - Human primary fibroblasts, human primary keratinocytes, primary human endothelial cells (HuVEC), human skeletal muscle cells, HeLa cells and C2C12 mouse myoblasts were plated in 24-well plates in complete medium without antibiotics at the optimal plating density for each cell type to yield 50-70% confluency at the time of transfection. Cells were transfected with the pSpCas9(BB) -2A-GFP plasmid (9.3kb )(source: Addgene), using DNA-In® CRISPR reagent and incubated overnight at 37°C. Cells were then fixed and stained with anti-GFP and anti-Cas9 antibodies to better observe the transfection efficiency.
EditPro™ Transfection Reagent has been optimized to deliver Cas9 mRNA or RNP into a wide range of adherent cell types. It is well suited for delivery of gene editing tools, therefore, into many primary cells and cell lines.
The table below illustrates the optimal uses of our different reagents that may be used for gene editing. Click to the product pages for additional information and data:
Genome Editing Reagents
|mRNA||Protein (RNP)||Cell Type|
|XX||X||XX||XX||huES, iPSC, NSC|
|XX||XX||adherent primary cells and cell lines|
|XX||XX|| primary fibroblasts, muscles cells, keratinocytes and other adherent primary cells |