TALENs—an indispensable tool in the era of CRISPR: a mini review part 1

REVIEW Open Access TALENs—an indispensable tool in the era of CRISPR: a mini review Anuradha Bhardwaj and Vikrant Nain* Abstract Background: Genome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs. Main body: Genome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs. Conclusions: This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing. Keywords: Genome editing, TALEN, CRISPR, ZFN, Meganuclease Background Genome editing Genome editing is a procedure that allows for site- specific modifications to be made in the genome of any organism [1]. There are two main goals for genome ma- nipulation: one is to learn how new genes function and what functions they play in cell regulation. The second major use of genome manipulation is the creation of al- ternative treatment options for a variety of genetic disorders [2]. The evolution of genome engineering using designer nucleases has brought a paradigm shift in the field of biotechnology. These tools have demonstrated promising results in various domains including medical and agricultural sciences [3]. Also, both TALENs and CRISPR are crucial in elimination of undesired genes [4]. The appealing agricultural applications of nuclease-based genome editing include improved varieties of crops with high yields and desired features like enhanced nutritional content, greater shelf life, better stress tolerance, disease, and pest resistance [5]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. * Correspondence: vikrant.nain@gmail.com Department of Biotechnology, Gautam Buddha University, Greater Noida, Uttar Pradesh 201312, India Journal of Genetic Engineering and Biotechnology Bhardwaj and Nain Journal of Genetic Engineering and Biotechnology (2021) 19:125 https://doi.org/10.1186/s43141-021-00225-z.

[Audio] Double-strand breaks in DNA are particularly deleterious, but all living species have evolved repair mechanisms to restore the initial sequence in order to protect the functionality of their genomes. As soon as the double-strand break is created, either of the DNA repair pathways gets activated. There are two main mechanisms: nonhomologous end joining and homologous recombination. Nonhomologous end joining is preferred for gain/loss of function applications because of its mutagenic behavior, whereas homologous recombination is cell cycle-dependent and has reduced efficiency due to its dependence on a template. Both mechanisms are extensively employed to manipulate genes..

[Audio] Both TALENs and CRISPRs induce a double-strand break on the target DNA, leading to the activation of different cell repair pathways. However, while TALENs recognize the target site based on DNA-protein interaction, CRISPRs rely on site-specific RNA-protein interactions. This fundamental structural difference between TALENs and CRISPRs leads to their strengths and weaknesses in terms of designing, synthesis, efficiency, specificity, and off-target activity..

[Audio] The CRISPR-Cas9 system has been widely used in various fields, including genetic engineering, biotechnology, and medicine. The system consists of two main components: the Cas9 enzyme and a guide RNA (gRNA). The gRNA is programmed to recognize a specific target sequence, and the Cas9 enzyme cuts the DNA at that site. The system has been used to edit genes, silence gene expression, and even introduce new genes into cells. In addition, the CRISPR-Cas9 system has been used to treat genetic diseases, such as sickle cell anemia and muscular dystrophy. The system has also been used to develop new crops and improve crop yields. Furthermore, the CRISPR-Cas9 system has been used to study the epigenetic regulation of gene expression and to understand the mechanisms of gene silencing..

[Audio] The unique features and advantages of TALENs as a genome editing tool include their specificity, low cytotoxicity, and ability to target hyper variable sites. They consist of 18 repeats of 34 amino acids and require binding to the target site on opposite sides with a spacer of 1420 nucleotides in between. Their degenerate codes allow them to bind to multiple nucleotides with varying levels of efficiency, making them a more flexible and reliable tool for genome editing. Additionally, TALENs are the only known genome editing tool that can be engineered to escape mutations in a genome, making them highly applicable for clinical use. Their high level of specificity is particularly important when targeting specific genes within a gene family or a particular allele in a polyploid plant species..

[Audio] The TALENs, or Transcription Activator-Like Effector Nucleases, have demonstrated a wide range of applications in modulating gene expression in various organisms. They can be easily re-engineered for specific DNA targets. While CRISPR has gained a lot of attention for its simplicity and efficiency, TALENs have several advantages that make them more suitable for clinical applications. Notably, TALENs can be re-engineered to be sensitive to DNA chemical modifications, such as methylation. This is due to the high structural resemblance of methylated cytosine to thymine, allowing for efficient binding by TALENs' non-canonical RVDs. Moreover, TALENs also exhibit a unique characteristic of degeneracy - where a single RVD can code for multiple epigenetic nucleobases, such as 5mC and 5hmC. This enables TALENs to directly distinguish between methylated and unmethylated cytosines, making them a powerful and versatile tool for genome editing. In terms of transcription activation, TALENs have a clear advantage over CRISPR. Artificial transcription factors were first developed by fusing a zinc finger protein with a transactivation domain from the herpes simplex virus. However, the large size of the complex makes it less efficient compared to TALEN protein-based artificial transcription factors..

[Audio] The recent study by Bhardwaj and Nain (2021) highlights the effectiveness of TALENs in editing the epigenome using KRAB, Sid4, or SRDX repressors. TALE-proteins have emerged as a preferred DNA-binding scaffold due to their simple cipher and compatibility with various epigenetic modifiers. This enables precise targeting of epigenetic effector domains to any locus in the genome, revealing the exact sequence of chromatin remodeling events. TALENs have shown compatibility with viral vectors like AAV, AdV, and LV, making them a promising option for in vivo delivery. They offer a promising alternative for clinical applications due to their simplicity and broad range of epigenetic modifier compatibility..

[Audio] The unique features and advantages of TALENs over CRISPR include their higher efficiency and specificity, precision in targeting, longer target recognition site, and reduced risk of off-target effects. These characteristics make TALENs a more suitable option for clinical applications, especially considering the limitations and ethical considerations surrounding CRISPR..