Assessment of the Feasibility of TALEN

Abstract

DNA, otherwise known as deoxyribonucleic acid, is an essential part of every living organism. However, with hereditary diseases such as cystic fibrosis and diabetes, is there any method to remove malign traits? In this article, a gene-altering tool is explored to provide readers with a general understanding as well as the practicability of this instrument. The main section of this article focuses on the advantages and disadvantages of this instrument, which will be used to determine the usability of this technology. Additionally, this article is unique through the comparison of multiple gene-editing tools and the discussion of ethical aspects of gene-editing.

Introduction

The action of suppressing or removing a segment of DNA has been practiced in many laboratories around twenty years ago. However, the usage of gene-editing tools has recently been on the rise.^[1] Due to its strong potential to cure hereditary diseases, all risks and gains must be accounted for, including ethical concerns that have been raised by society. In response to this dilemma, this article aims to reach a decisive conclusion as to whether gene-editing tools should be used on humans.

What is TALEN?

TALEN, otherwise known as transcription activator-like effector nuclease, is used to cut or remove targeted DNA strands. In order to produce a TALEN, a TAL effector must fuse with a DNA cleavage domain, which is the nuclease that cuts the DNA strand. A TALEN can be engineered to attack a specific sequence in the DNA, thus allowing the instrument to target specific genes or traits.^[2] Once the DNA strand is cut, the fixed DNA is introduced in order to replace the faulty DNA strand. Once this process is complete, the cell finalizes the DNA strand through a procedure known as homologous recombination. Additionally, a TALEN can knock out a gene, which means that the trait that the gene possesses is entirely removed from the DNA.

Figure 1: Two Methods of Gene Knockouts with TALEN ^[3]

Discussion (Advantages and Disadvantages)

Due to its strong potential, TALEN has many advantages to offer. Its long 33-35 amino acid chain can target specific nucleotides, which increases the accuracy in finding the correct DNA strand. However, due to the complexity of the amino acid chain, the gene-editing instrument requires a large number of resources to create. One method to produce TALEN is through commercial synthesis. Unfortunately, as Tomas Cermak of the University of Minnesota states in his nucleic acid research, commercial synthesis is an expensive process that reduces accessibility.^[4]
Additionally, TALEN can be used on livestock. Since DNA is made of the same base nucleotides in every living organism, TALEN has the possibility of editing genes outside of human DNA. As Daniel F. Carlson concludes in his TALEN efficiency in livestock research, TALEN had completed 75% of gene knockouts within the embryos included in the experiment.^[5] The implication of gene editing in livestock is significant because Carlson finds that gene knockouts can increase their resistance to certain diseases as well as produce models that simulate human diseases.
Moreover, TALEN has been known to induce more mutations when compared to another gene-editing instrument, ZFN (Zinc finger Nucleases). According to research conducted by Shijia Chen, TALEN is more likely to induce up to 10 times as many mutations when compared to ZFN.^[6] This finding helped Chen reach her conclusion, stating that TALEN is the superior technology to use for gene editing and targeted mutations.
TALEN also has numerous business applications. Richard Hamermesh, the faculty co-chair of the Harvard Business School/Kraft Precision Medicine Accelerator, stated that current medical companies are seeking interest in areas of gene therapies that have the potential to stop diseases that have no cure.^[7] Since TALEN has the potential to eradicate single-gene diseases, pharmaceutical companies will compete to produce this treatment, which increases accessibility to the public.
However, no instrument is flawless, as there are limitations and drawbacks of TALEN. An inherent disadvantage of the TALEN is that it only can help prevent hereditary diseases that affect one gene. Examples of these diseases include sickle cell anemia and cystic fibrosis.^[8] Another issue with TALEN is that the controlled mutations can result in severe complications for the patient. Robert Truog, director of the Center for Bioethics at Harvard Medical School, corroborates the danger of TALEN as he states that “random mutations often cause serious problems, and people are born with serious defects.” ^[9]
Due to improved traits or prevented illnesses that occur as a result of TALEN, an ethical dilemma occurs. Since TALEN is an expensive treatment, individuals worry that this technology will only be available to the wealthy and upper class, placing a significant disadvantage to the rest of society. Additionally, using gene-editing instruments can pose a serious risk to the patient as there is always a chance for off-targets to occur, thus interfering with the patient’s DNA. One example of this situation occurred in 2018.
He Jiankui, a gene-editing researcher, presented his research in 2018, where he stated that he had produced the world’s first gene-edited babies.^[10] However, both the public and the scientific community responded with anger. Jiankui used gene-editing to deactivate a gene known as CCR5, a common entrance for HIV to attack immune cells. However, deactivating CCR5 was entirely unnecessary as both babies did not carry HIV and there is already a medication that can deactivate CCR5 and has been clinically tested. .^[11] Thus, the use of TALEN is not widespread, and even in the hands of scientists, errors can occur.

Comparison to Other Instruments

Within the area of gene-editing, there are two other instruments that scientists use to induce controlled mutations: ZFN and CRISPR-Cas9. ZFN, otherwise known as Zinc-Finger Nuclease, uses a DNA-binding protein as well as a DNA cleavage domain that targets a specific genome within the DNA and executes a double-strand break. ZFN and TALEN are quite similar in terms of structure, as both instruments contain a DNA cleavage domain. However, in terms of performance, TALEN has a much stronger mutation rate. This high mutation rate is due to the fact that TALEN and ZFN have different anchor sites. ZFNs are able to recognize nucleotides in groups of three, whereas TALENs are able to recognize individual nucleotides.^[12] Thus, due to the lack of recognition within ZFN, they are more prone to off-target sites, which can cause more injuries to the patient.

Figure 2: (A) represents ZFN recognizing nucleotides in groups of three, while TALEN recognizes individual nucleotides ^[13]

CRISPR-Cas9, otherwise known as Clustered Regularly Interspaced Short Palindromic Repeats, is another gene-editing tool that uses a guide RNA to find the targeted DNA strand. The Cas9 protein then performs the double-stranded cut, leaving that cell to introduce the mutation. One advantage that sets CRISPR-Cas9 out from the rest of the gene-editing instruments is its efficiency. CRISPR-Cas9 can be injected directly into the embryo, which reduces the amount of time required to perform the gene edit. However, in comparison to TALEN, CRISPR-Cas9 has performed many off-targets. Brandon Specktor of Live Science states that while CRISPR-Cas9 has executed the desired gene edit, it has interfered without cells and removed essential DNA. Specktor concludes that this loss of essential DNA occurred in about 15% of cases.^[14]

Conclusion

With numerous advantages and comparable disadvantages, the usability of TALEN is questionable. Numerous factors can go wrong, especially when the instrument leaves the environment in order for the cell to induce the mutation. Although TALEN is not as efficient compared to other instruments such as CRISPR-Cas9, it can handle gene-edits reliably. However, without the approval of both the public and the scientific community, it is unethical to even perform such operations. Nonetheless, gene-editing instruments have underlying factors such as costs that decrease the accessibility to this technology. Thus, TALEN is a viable option whose usability should be decided on a case-by-case basis.

References

1. American Chemical Society. “The rise, fall and resurgence of gene therapy.” ACS News Service Weekly PressPac (2019): e20-20.
2. Addgene. “Addgene\’s Guide to TALEN Technologies.” Addgene December Newsletter (2011): e11-e11.
3. Ed Davis. “Genome Editing: Which Should I Choose, TALEN or CRISPR.” GeneCopoeia (2014).
4. Cermak, Tomas, Erin L. Doyle, Michelle Christian, Li Wang, Yong Zhang, Clarice Schmidt, Joshua A. Baller, Nikunj V. Somia, Adam J. Bogdanove, and Daniel F. Voytas. \”Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.\” Nucleic acids research 39, no. 12 (2011): e82-e82.
5. Daniel F. Carlson, Wenfang Tan, Simon G. Lillico, Dana Stverakova, Chris Proudfoot, Michelle Christian, Daniel F. Voytas, Charles R. Long, C. Bruce A. Whitelaw, and Scott C. Fahrenkrug. “Efficient TALEN-mediated gene knockout in livestock.” October Newsletter, vol. 109, no. 43 (2012).
6. Shijia Chen, Grigorios Oikonomou, Cindy N. Chiu, Brett J. Niles, Justin Liu, Daniel A. Lee, Igor Antoshechkin, David A. Prober. “A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly.” Nucleic Acids Research 4, vol. 39, issue 4 (2013): e2769-e2778.
7. Mary Todd Bergman. “Perspectives on Gene Editing.” Harvard Gazette (2019).
8. Dr. Stacey Wirt. “Editing our DNA with Molecular Scissors.” Tech Interactive.
9. Mary Todd Bergman. “Perspectives on Gene Editing.” Harvard Gazette (2019).
10. Jon Cohen. “The untold story of the ‘circle of trust’ behind the world’s first gene-edited babies.” AAAS Science (2019).
11. Ed Yong. “The CRISPR Baby Scandal Gets Worse by the Day.” Atlantic Science (2018).
12. Nathan Packel. “Comparison Between ZFN, TALEN and CRISPR.” Nuclineer’s Genetics (2019).
13. Addgene. “Addgene\’s Guide to TALEN Technologies.” Addgene December Newsletter (2011): e11-e11.
14. Brandon Specktor. “CRISPR Gene Editing May Be Doing More Damage Than Scientists Thought.” Live Science News (2018).

About the Author

Tony Joseph is a Year 10 student at Walt Whitman High School, located at Bethesda, Maryland, in the United States. Tony is currently pursuing a passion for biology and hopes to major in this degree at college, which will lead him to a career in medicine.