Introduction
Ever since the discovery of the DNA, several technologies have evolved to genetically manipulate the organisms for better agricultural and industrial applications. The next generation sequencing techniques have made the identification of the different genes and mutations in DNA easier. The GMOs and cloning techniques which are in use for a significant time now are the direct result of the advancement in this domain. These cloning techniques have been exploited for a wide array of medical, agricultural, industrial and research applications for the past several decades. However, despite all the development and progress there is a constant need for more advanced gene editing techniques that are precise and accurate. The use of programmable nucleases for genetic modifications has increased in the past decade for its potential to treat various inheritable diseases and cancer. In past decade three major classes of programmable nucleases have come to light– the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)–associated Cas9 (CRISPR/Cas9).
Zinc finger nucleases (ZFNs)
ZFNs was the earliest developed genome editing tool with greater precision for their ability to alter the genetic content. The ZFNs are made of two domains, repeated zinc finger proteins (ZFPs) with their DNA-binding domains fused to the non-specific DNA cleavage domain of a FokI restriction endonuclease. ZFNs function as a dimer where each monomeric unit has an ability to recognize nine to 18 base pairs (bps) of DNA via the zinc-finger DNA-binding domain. There is a wide range of Zinc finger domains which recognize the distinct DNA triplets. These can be joined together to generate polydactyl zinc-finger proteins that can target a wide range of possible DNA sequences. However, the off-target editing/mutations are the major concern associated with the ZFNs based gene editing. Therefore, there is ample scope for enhancing the efficiency of this process.
Transcription activator-like effector nucleases (TALENs)
TALENs has emerged as an alternative to ZFNs for genome editing. Similar to the ZFNs, TALENs comprise two programmable DNA-binding domains fused to a FokI endonuclease domain and works by introducing the double-strand breaks (DSBs). Transcription activator-like effectors (TALEs) are naturally secreted by the Xanthomonas spp. of bacteria which binds to the DNA of the host organism. The TALE DNA binding domain is multiple repeats having 33-35 amino acids where each repeat recognizes a single nucleotide. The specificity in the TALE DNA binding domain is due to the presence of hyper variable regions present in each domain at 12 and 13 amino acid positions. Therefore the sequence specific TALENs can be generated by modifying these amino acid residues in hyper variable regions and linking the different TALE repeats together.
Clustered regularly interspaced short palindromic repeats (CRISPR)–associated Cas9 (CRISPR/Cas9)
In addition to ZFNs and TALENs, the CRISPR/Cas9 is another rapidly emerging gene editing tool. It is a naturally occurring bacterial system which acts as a component of bacterial adaptive immunity. It provides protection to bacteria from invading viruses and plasmids via RNA-guided DNA cleavage by Cas protein. There are three main components of the CRISPR/ Cas9 system–the CRISPR RNA (crRNA), the trans-activating crRNA (tracrRNA) and the Cas9 enzyme. The crRNA is transcribed from the invading system and known as protospacer sequence and hybridizes with the tracrRNA that triggers and acts as base for the binding of Cas9 nuclease to the site of DNA cleavage. Unlike ZFNs and TALENs systems that recognize the specific sequences in the given genome sequence using different recognition proteins, the CRISPR/Cas system uses the RNA sequence complementary to the target genomic sequence. The advantage of the RNA hybridization in site specific cleavage triggers the use of chimeric “guide” RNA (gRNA) for the genomic editing, which is created by fusing together the crRNA and tracrRNA. The gRNA contains 20 nucleotide guide sequences designed for binding to specific DNA sequences. Therefore, the gRNA and Cas9 are able to scan the appropriate site and bind to it for creating site specific double strand breaks (DSB). Thus, the CRISPR/Cas9 system offers a relatively more accurate technique of gene editing as compared to the previous techniques and can be used for a wide range of applications.
Conclusion
So far, it is evident that the current gene editing mechanisms are more precise and possess a wide range of application without having greater ethical and physiological implications. Therefore, greater financial advantages and continuous improvements in this field are inevitable. Also, rational research and development, proper target identifications and intellectual property protection is significantly important for developing the next generation therapeutics, biological products and processes.
