Recent exciting advances in gene editing technologies such as clustered regularly-interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins or transcription activator-like effector nucleases (TALENs) have made it faster and simpler for researchers to create targeted, sequence-specific cuts in DNA at an area of interest.
By providing donor DNA with homologous ends matching the ends of the induced DNA breaks in the cellular genome, a scientist may then utilize cells’ natural DNA repair mechanisms to stitch DNA together in new ways. Designer cells may then be created with customized DNA sequences. Advances in gene-editing techniques are making it more efficient to make small changes in a single target gene, to knock-in or replace whole genes or to edit multiple different genes at the same time in a single step.
Future applications for these gene editing technologies are only limited by a researcher’s imagination and their ability to deliver these tools to the target cells or tissues. Simple point mutations can be created in easy-to-transfect cells in vitro by delivering editing constructs with traditional transfection methods. However, for more difficult to transfect samples such as primary cells, 3D cultured cell structures or tissues in vivo, more efficient delivery methods are required. Similarly, for more complex insertions, gene replacements or multiplexed gene editing experiments where large or multiple constructs are being delivered, improving transfection efficiency is critical to success.
Electroporation as a transfection method offers the advantage of facilitating the efficient delivery of large and complex payloads in a universal manner to all cell and tissue types. In this article, the principles of electroporation as a transfection method will be reviewed, some of the chief enhancements in commercially available electroporation instruments and electrodes will be discussed and examples of CRISPR gene editing techniques made possible by electroporation methods will be provided to illustrate the potential for research and discoveries that could be made as a result of this work in 2019 and beyond.
Electroporation is a physical transfection method that works by passing an electrical current through a cell or tissue sample. The current delivered by electroporation instruments induces charge differentials between the inside and outside of the cell membranes, resulting in the formation of transient pores in the cell membrane. During this carefully controlled electrical pulse, the gene editing constructs move into the cell through these pores and the cell membrane reseals after the completion of the pulse. With electroporation, the size of the gene editing constructs and donor knock-in DNA molecules that are delivered to the target cells are not limited. Electroporation may also penetrate to transfect cultured organs or whole tissues of live organisms.
Currently available electroporation equipment is tailored to fit different types of applications depending on the researcher’s target cell or tissue types. Different types of waveforms, or electrical pulse shapes, are available to target different types of samples. Square electroporation waves consist of pulses that are delivered at a constant voltage, whereas exponential decay waves consist of pulses that begin at a peak voltage which then decays exponentially over time. Square wave electroporation systems such as the BTX ECM 830 are ideal for transfecting mammalian cells and tissues. Exponential decay wave systems such as the BTX ECM 630 are ideal for transforming bacteria, yeast and other microorganisms.
For laboratories that need to universally transfect or transform all cell types of organisms, instruments that offer both square and exponential decay waveforms such as the BTX Gemini Twin Wave Electroporator offer both square and exponential decay modes for universal electroporation of any sample. In addition, electrofusion equipment such as the BTX ECM 2001+ offer more complex combinations of alternating current and square waveforms that may be employed in cell fusion and embryo manipulation. These types of instruments are best suited for applications utilizing gene editing technology to create transgenic research animals.
The electrodes utilized in electroporation are also customizable to fit experimental needs. For cells in vitro, the cells are removed from the culture vessel and suspended in an electroporation buffer containing the cargo molecules of interest. Then the cell and transfectant suspension is placed in contact with electrodes—typically in a chamber such as an electroporation cuvette—and connected to an electroporation generator that passes the current through the sample. For high-throughput applications, multi-well electroporation plates can transfect 5 to 384 samples at a time. Other types of electrodes are designed to sit inside culture vessels to transfect cells in an adherent state. For 3D cultured cells or slices of tissues in vitro or in vivo, the sample may be placed into a specialized electrode chamber, contacted with paddle-shaped electrode probes or reached by electrode needles inserted into the tissue.
One key benefit of electroporation as a means to facilitate gene editing technology is its efficient transfection of large molecules. Traditional chemical and lipid transfection methods are limited in efficiency for gene knock-ins. As an example of this, Fenghua Lu, et al.1 compared the efficiency of lipid transfection reagents and electroporation as methods to transfect gene editing constructs for knock-in of an enhanced green fluorescent protein (EGFP) reporter gene into buffalo fetal fibroblasts (BFFs), which were subsequently used to create transgenic GFP-expressing animals. Electroporation was found to have higher transfection efficiency than both lipid reagents when assessed both at the stages of producing knock-in BFFs and in successful transgenic embryo development to the blastocyst stage. Ultimately, researchers were successful in creating live births of transgenic EGFP-expressing calves.
A second important way that electroporation is enabling more advanced gene editing technologies is its ability to efficiently penetrate 3D structures, transfecting tissue in a way that chemical, lipid and viral delivery methods cannot. Weijun Feng et al.2 and Xuan Yao et al.3 have utilized an in-utero electroporation strategy to deliver gene editing constructs to study the development of the fetal brain in vivo.
Third, electroporation simplifies the workflow for the researcher and ensures greater reliability of results. A number of electroporation instruments available today offer preset starting electroporation parameters for common sample types to reduce the amount of optimization on the researcher’s part. Many current instruments also offer storage of custom user protocol parameters as well for push-button convenience. Electrical pulse and sample resistance monitoring and data logging features ensure consistency from experiment to experiment. The simplicity of electroporation instrumentation and methodology was harnessed by Wenning Qin et al.4 to develop in vivo electroporation of early-stage embryos with gene editing nuclease constructs. This method, named zygote electroporation of nuclease (ZEN), offers an easier alternative to microinjection techniques that require expensive microscopy and micromanipulator equipment and a much greater degree of technical expertise.
In summary, electroporation instrumentation and techniques are increasingly becoming the methods of choice for easier, faster and more efficient delivery of molecules of interest to more biologically relevant target model systems. Electroporation boosts the efficiency of gene editing tools, enabling researchers toward the next step of innovation and discovery.
Michelle M. Ng, PhD is the Global Product Manager of the BTX line of electroporation and electrofusion products and the QuikPrep line of Sample Preparation Products at Harvard Bioscience in Holliston, Massachusetts.