Genome editing in crop improvement: Present scenario and future prospects

ABSTRACT Genome editing refers to a process by which a specific chromosomal sequence is changed. The edited chromosomal sequence may comprise an insertion of at least one nucleotide, a deletion of at least one nucleotide, and/or a substitution of at least one nucleotide. Genome editing is a relatively new technology that is gaining importance as a tool for crop improvement because of its advantages over routinely used methods of genetic engineering. Genome-editing technology is precise and efficient. Genome editing is now considered a safe technique because no foreign sequences are left behind in the final genome-edited organism (GEO). Genome editing involves the induction of double-stranded breaks (DSBs) at specific sites of DNA, which turns on endogenous repair mechanisms—homology-dependent repair (HDR)—when homologous sequences are present, and nonhomologous end-joining (NHEJ) in the absence of homologous sequences. During repair, site-specific mutations are produced. A range of molecular tools for inducing DSBs at specific sites of a genome is available with genome editors. One category of such molecular scissors include engineered and programmable site-specific nucleases (SSNs), such as meganucleases (MNs), also known as homing nucleases (HNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nuclease (RGN) systems, the most widely used RGN being the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system 9 (CRISPR/Cas9), and DNA-guided nuclease (DGN) system, i.e., NgAgo (an acronym for Natronobacterium gregoryi Argonaute). Transposons and Group II intron retro-transposition have also been employed in genome editing. Some new genome-editing approaches have also emerged under the umbrella of triplex technology, which are based on antisense technology and make use of diverse types of oligonucleotide-linked nucleases, triplex-forming oligonucleotides, nucleic acid analogs, peptide nucleic acids, and aptamers for providing homologous sequences for HDR. Some engineered animal viruses such as lentiviruses, adeno-associated viruses, recombinant adeno-associated viruses, and adenoviruses (AdVs) and plant viruses such as RNA viruses, tobacco rattle virus (TRV), and single-stranded DNA (ssDNA) viruses called geminiviruses have been used as genome-editing devices that act as delivery vehicles of SSNs. Easy programmability of CRISPR/Cas9 and the development of its modified versions have shown versatility in their functions and have been successfully applied in yeast, animals, human and nonhuman cell lines, and embryos for gene therapy. The CRISPR/Cas-produced gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. The NgAgo is still a developing technology although it has shown promise and some advantages over CRISPR/Cas systems. However, CRISPR/Cas systems still dominate the plant genome-editing scenario. This is the reason that the present review deals in detail with various aspects of CRISPR/Cas systems but briefly with other genome-editing tools. Safety, legal, intellectual property (IP), and regulatory issues need to be addressed to the satisfaction of scientists, farmers, and consumers to fully exploit the potentials of different platforms of genome-editing technology in crop improvement and other areas such as animal improvement, microbial engineering, and gene therapy.