Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review

Abstract

The emergence of genome-editing technology has allowed manipulation of DNA sequences in genomes to precisely remove or replace specific sequences in organisms resulting in targeted mutations. In plants, genome editing is an attractive method to alter gene functions to generate improved crop varieties. Genome editing is thought to be simple to use and has a lower risk of off-target effects compared to classical mutation breeding. Furthermore, genome-editing technology tools can also be applied directly to crops that contain complex genomes and/or are not easily bred using traditional methods. Currently, highly versatile genome-editing tools for precise and predictable editing of almost any locus in the plant genome make it possible to extend the range of application, including functional genomics research and molecular crop breeding. Vegetables are essential nutrient sources for humans and provide vitamins, minerals, and fiber to diets, thereby contributing to human health. In this review, we provide an overview of the brief history of genome-editing technologies and the components of genome-editing tool boxes, and illustrate basic modes of operation in representative systems. We describe the current and potential practical application of genome editing for the development of improved nutritious vegetables and present several case studies demonstrating the potential of the technology. Finally, we highlight future directions and challenges in applying genome-editing systems to vegetable crops for research and product development.

Keywords: CRISPR-Cas application; genome-editing technology; precision breeding; transformation; vegetables.

Figure 1

Some of the major genome-editing technologies using site-specific nucleases include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9) systems. (A) The ZFN-binding domain is comprised of multimerized zinc-finger proteins (ZFPs). Each ZFP recognizes approximately 3 bp of DNA sequence, and the fused FokI nuclease domains dimerize and generate double-strand breaks. (B) Like ZFNs, TALENs consist of a DNA-binding domain, termed transcription activator-like effector (TALE) repeats, and nuclease domain of FokI enzyme. Each TALE repeat consists of a highly conserved 34-amino acid sequence with hypervariable twelfth and thirteenth amino acids, which allow the recognition of the single nucleotide. (C) In the CRISPR-Cas9 system, a single-guide RNA (sgRNA) guides the Cas9 nuclease to direct the cleavage of cognate DNA sequences adjacent to 5'-NGG-3' protospacer-adjacent motifs (PAMs).

Figure 2

The main CRISPR-Cas-mediated genome-editing systems. (A) Diagram of the CRISPR-Cas9 is shown with a sgRNA encoding a spacer guide RNA (gRNA) positioned next to PAM (5'-NGG) site. Cleavage sites by Cas9 protein are shown with scissors, and blunt ends are presented with dotted line. (B) A diagram of the CRISPR-Cpf1 is shown with crRNA encoding a spacer gRNA positioned next to PAM (5'-TTTTN) site. Cleavage sites by Cpf1 protein are shown with scissors, and staggered ends with 5' overhang are presented with dotted line. (C) Base editor (BE) composed of nCas9 nickase (D10A). The base editing system has two versions: adenine and cytidine BEs converting A to G and C to T, respectively. (D) nCas9 nickase (D10A) fused with adenine base editor and cytidine base editor generates A to G and C to T substitutions, simultaneously. (E) Prime editor composed of nCa9 nickase (H840A), reverse transcriptase (RT), and prime editing guide RNA (pegRNA). The pegRNA carries spacer, desired editing sequence, and primer binding site (PBS). The PBS binds to the nicked strand, and then, RT copies sequences from the template. Flap equilibration results in unchanged and mutated DNA strands.

Figure 3

Strategies for delivery of the CRISPR-Cas system into plants. (A) The preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) can be delivered into protoplasts through the polyethylene glycol (PEG)-mediation, and T-DNA encoding CRISPR-Cas reagents [Cas protein and sgRNA(s)] can be delivered into the rigid plan cells (explants, microspores/pollens, and intact plants) using Agrobacterium-mediated transformation, biolistic bombardment, and magnetofection. Subsequently, regeneration procedures of protoplasts and tissues carrying CRISPR-Cas reagents are needed to produce genome edited lines. (B) In the virus-induced gene-editing system, sgRNA fused with RNA mobile element is integrated into tobacco rattle virus (TRV) RNA2. After transformation of TRV RNA1 and TRV RNA2 to Agrobacterium, infiltration is conducted to Cas9-overexpressing plants resulting in systemic spreading of the sgRNA by the mobile elements and induction of mutagenesis. (C) For de novo meristem induction system, the meristems of Cas9-overexpressing plants are removed for infiltration, and then, Agrobacteriumcarrying morphogenic regulators (MRs) and sgRNA are injected into pruning sites. MRs induce the de novo gene-edited meristem, and the gene-edited plants can finally be obtained from newly developed shoots.