Jon Chesnut Senior Director Thermo Fisher Scientific

Breaking Down the Differences between the Two Best Gene-Editing Tools on the Market

Genome editing can be defined as specifically and surgically knocking out genes or altering the DNA sequence of a cell precisely in order to modify gene function. As convincingly shown by Mario Capecchi in 1986 with the advent of targeted gene modification in mice, this process— which relies on the cellular machinery to incorporate a DNA “donor” by homologous recombination—is powerful but also inherently inefficient. It was subsequently discovered that efficiency could be vastly improved by the introduction of a single- or double-stranded break in the genome near the site of the intended edit. Since then, improvements in genome (or gene) editing have largely been driven by the discovery of better tools with which to introduce that targeted break. Fast forward to today and the CRISPR revolution.

With knowledge of its existence expanding beyond scientific circles, CRISPR has popularized the concept that it may soon be possible to precisely and elegantly—and almost routinely—modify DNA to accomplish incredible things. Applications range from the relatively “straightforward” (simplifying the generation of cell models of disease) to the cutting edge (a new generation of gene and cell therapies). Beyond the biomedical applications lies a whole host of other opportunities, for example, agriculture and metabolic engineering of industrial organisms.

The key to high efficiency gene editing is to create a targeted break in the genome as specifically and efficiently as possible. Irrespective of other steps in the process, this step greatly impacts the frequency of correctly edited cells within the overall pool of cells where the attempt was made. Several tools capable of generating the DNA break have been developed over the years. Currently, the two best tools are CRISPR and TALENs. While CRISPR is often the tool of choice for many of these applications, TALENs can be just as effective, in many cases providing a better solution to the problem at hand. 


The TALEN Advantage: Ultra Precision with A Clear Path Toward Complete Freedom to Design and Operate

TALENs, or transcription activator-like effector nucleases, are a widely used technology for precise and efficient gene editing in live cells. They are a particularly useful tool for gene knock-ins, where precise targeting is critical. Initially limited to target sequences where each TALE protein monomer must bind a 5’ thymidine, TALENs are now engineered to be positioned in the genome without restriction. CRISPR, on the other hand, requires a three base PAM sequence (NGG preferred) immediately after the gRNA target sequence on the DNA. When editing by inserting donor DNA, this positional limitation can result in substantially reduced homologous recombination efficiency depending on the distance from the PAM to the intended edit.

When it comes to specificity, researchers theorize that TALENs may result in fewer off-target cleavage events, thereby reducing undesirable mutations within the cell. This is a potential advantage in clinical applications of TALEN-edited cells, such as the recent use of allogeneic engineered T cell (CAR-T) therapy to successfully treat two young children suffering from acute lymphoblastic leukemia.

TALEN technology also offers a significant advantage for organizations requiring a clear licensing path for commercial applications. While the CRISPR IP landscape has not yet been settled, two companies— Thermo Fisher Scientific and Cellectis—control and cross-license foundational IP in the TAL Effector space. Their alliance, which includes the Two Blades Foundation for commercial applications in plants, clarifies the use of TALENTM TAL Nucleases for gene editing in research and applied markets.


Where CRISPR Shines: Simplicity, High Cleavage Efficiencies and Versatility

With their ease of design and robust cleavage activity, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. Particularly for academic researchers, CRISPR has served as an accessible alternative to TALENs, which traditionally take more time to create and can be more expensive. Overall, CRISPR has higher expected cleavage efficiency than TALENs— a substantial advantage for many R&D applications. CRISPR also offers versatility. Since CRISPR’s nuclease (Cas9) is constant, the protein can be bulk manufactured and the small guide RNA, which lends specificity, can be designed and produced rapidly and in high throughput. The two can then be efficiently delivered to cells as a purified Cas9/gRNA complex at a controlled dose.

As screening and gene editing expands to more relevant and harder to transfect cells, lentivirus-based CRISPR platforms can improve delivery and lead to more effective genome modification. Pharmaceutical companies are now using lentiviral-based CRISPR libraries for high-throughput applications of functional genomics screening. This new approach to drug discovery, which likely will be at least a companion to RNAi screening and could emerge as the “go to” technology in this space, has the potential to provide breakthroughs in research by targeting and discovering genes involved in the development of diseases such as cancer. Additionally, several clinical trials have recently been proposed based on CRISPR-edited cells: Modified T-cells with increased efficacy against certain cancers (U Penn), treatment of a rare form of blindness, Leber congenital amaurosis (Editas Medicine) and others (CRISPR Therapeutics and Intellia Therapeutics).


Conclusion

There is no one right answer when it comes to choosing CRISPR or TALENs— the best gene-editing tool depends on the application. Determining the optimal tool starts with understanding the cell lines you’ll be working with and your end research goals. In general, CRISPR is the preferred tool for gene knock-out studies and screens. However, if specific gene editing (repairing or inserting a mutation for instance) is the goal, TALENs are an efficient option as they can create a cleavage site essentially anywhere in the genome where CRISPR cannot. Both TALENs and CRISPR can achieve high levels of cleavage efficiency in a variety of cell types and both are effective options for therapeutic applications where the time and cost of identifying a single highly active reagent is minimal relative to other development costs.

Technically speaking, these tools serve various niches in the genome editing and cell engineering continuum. This is true from a commercial angle as well. If the goal is to use genome editing or edited organisms in a commercial setting, one must keep in mind the IP landscape and should consider TALENs as a tool that can perform comparably with CRISPR and offer a clear path to commercialization.

Clearly we are living in exciting times. Our ability to sequence the genome is giving us a wealth of information on disease-related genetic mutations. Match this treasure trove with the revolution in efficient genome editing tools and one feels that we are at a tipping point for a new era of precision medicine. That said, it is important to keep in perspective that CRISPR as a broadly used genome-editing tool has only been around since 2012, and TALENs only a few years more. At this rate of development, it’s likely that we are only just beginning to realize the potential of genome editing for finding better drugs and as precision therapies in the clinic.







































Jon Chesnut is senior director of synthetic biology R&D at Thermo Fisher Scientific.

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