Lakhasly

Abstract Genome editing (GE) tools have been revolutionizing life sciences by various marvelous applications for targeted gene modifications in organisms. GenEd tools, for example, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems have been widely used in the last decade for genetic manipulation of plants, animals, microbes, and other organisms. The utility of the CRISPR/Cas tool is widespread compared to other contemporary tools due to its simplicity, efficiency, cost-effectiveness, and accuracy. Moreover, there are a number of variants in the CRISPR/Cas toolkit which increased its usefulness. Along with a number of benefits of CRISPR/Cas including the unique feature of multiplexing, the system has also some critical limitations and concerns. Off-targeting is one of the biggest limitations of the system which hinders its extensive use. There is a need to address the concerns associated with CRISPR to take more benefits from this system along with increasing biosafety and public acceptance. The future CRISPR system application in life sciences particularly human therapeutics and animal genome editing may be increased by mitigating the off- targets and other limitations of the system. The present review discusses gene editing techniques and outlines the application of the CRISPR system along with addressing its concerns and future prospects. Keywords: Genome editing – ZFNs – CRISPR - human genome - ethical issues 1. Introduction In classical genetics, the gene-modifying activities were carried out selecting genetic sites related to the breeder’s goal. Subsequently, scientists used radiation and chemical mutagens to increase the probability of genetic mutations in experimental organisms. Although these methods were useful, they were time-consuming and expensive. Contrary to this, reverse genetics goes in the opposite direction of the so-called forward genetic screens of classical genetics. Reverse genetics is a method in molecular genetics that is used to help understanding the function of a gene by analyzing the phenotypic effects of specific engineered gene sequences. Robb et al. (2019) defined and compared the three terms: “genome engineering”, “genome editing”, and “gene editing”. Genome engineering is the field in which the sequence of genomic DNA is designed and modified. Genome editing and gene editing are techniques for genome engineering that incorporate site-specific modifications into genomic DNA using DNA repair mechanisms. Gene editing differs from genome editing by dealing with only one gene (Khalil, 2020). The human genome developments paved the way for more extensive use of the reverse genetic analysis technique. Nowadays, two methods of gene editing exist: one is called “targeted gene replacement” to produce a local change in an existing gene sequence, usually without causing mutations. The other one involves more extensive changes in the natural genome of species in a subtler way (Laoharawee et al., 2018). In the field of targeted nucleases and their potential application to model and non-model organisms, there are four major mechanisms of site-specific genome editing that have paved the way for new medical and agricultural breakthroughs. In particular, meganucleases (MegNs), zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas-9). The aim of the present work is to discuss gene editing techniques and outline the .application of the CRISPR system along with addressing its concerns and future prospects