New breeding technique "genome editing" for crop improvement: applications, potentials and challenges

Abstract

Crop improvement is a continuous process in agriculture which ensures ample supply of food, fodder and fiber to burgeoning world population. Despite tremendous success in plant breeding and transgenesis to improve the yield-related traits, there have been several limitations primarily with the specificity in genetic modifications and incompatibility of host species. Because of this, new breeding techniques (NBTs) are gaining worldwide attention for crop improvement programs. Among the NBTs, genome editing (GE) using site-directed nucleases (SDNs) is an important and potential technique that overcomes limitations associated with classical breeding and transgenesis. These SDNs specifically target a compatible region in the gene/genome. The meganucleases (MgN), zinc finger nucleases (ZFN), transcription activator-like effectors nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonuclease (Cas) are being successfully employed for GE. These can be used for desired or targeted modifications of the native endogenous gene(s) or targeted insertion of cis/trans elements in the genomes of recipient organisms. Applications of these techniques appear to be endless ever since their discovery and several modifications in original technologies have further brought precision and accuracy in these methods. In this review, we present an overview of GE using SDNs with an emphasis on CRISPR/Cas system, their advantages, limitations and also practical considerations while designing experiments have been discussed. The review also emphasizes on the possible applications of CRISPR for improving economic traits in crop plants.

Keywords: Cluster regularly interspaced short palindromic repeats (CRISPR/Cas); New breeding technique (NBT); Site-directed nucleases (SDNs); Transcription activator-like effectors nucleases (TALENs); Zinc finger nucleases (ZFNs).

Fig. 1

Historical timeline of GE with respect to plants

Fig. 2

Types of different SDNs used for GE. a MgNs: schematic representation of naturally occurring I-SecI MgNs. Homing site for I-SecI is 18 bp and it cleaves DNA within the homing site. DNA binding and cleavage domains are not clearly demarcated. b ZFNs: schematic representation of synthetic ZFNs, ZFNs are synthesized by fusion of zinc finger DNA binding domain and FokI cleavage domain. Zinc finger DNA binding domains are typically composed of three zinc finger arrays each capable of recognizing approximately 3 bp. c TALENs: schematic representation of synthetic TALENs, TALENs are synthesized by fusion of TAL DNA binding domain and nonspecific FokI cleavage domain. Each TAL domain recognizes only one base. Binding specificity is manipulated by combining repeats that recognize individual bases in different orders. TALENs also work in dimer form. d CRISPR/Cas: schematic representation of CRISPR/Cas system. gRNA guides Cas9 protein for DNA DSB. gRNA form complex along with Cas9 protein and bind to seed sequence. Cas9 nucleases create DSB. Presence of PAM (NGG) sequence immediately downstream to target site is must for DSB

Fig. 3

Overview of various CRISPR/Cas 9-based applications. a Cas9 nucleases fused with activation domain can be used for transcriptional activation of targeted gene. b Cas9 nucleases fused with suppression domain can be used for transcriptional suppression of targeted gene. c Cas9 nucleases fused with chromatin modification enzyme DNMT (DNA methyl transferase) domain can be used for epigenetic modifications of DNA or histone. d Cas9 nucleases fused with GFP (green fluorescent protein) can be used to enable imaging of specific genomic locus