Genome-editing technologies and their potential application in horticultural crop breeding

Abstract

Plant breeding, one of the oldest agricultural activities, parallels human civilization. Many crops have been domesticated to satisfy human's food and aesthetical needs, including numerous specialty horticultural crops such as fruits, vegetables, ornamental flowers, shrubs, and trees. Crop varieties originated through selection during early human civilization. Other technologies, such as various forms of hybridization, mutation, and transgenics, have also been invented and applied to crop breeding over the past centuries. The progress made in these breeding technologies, especially the modern biotechnology-based breeding technologies, has had a great impact on crop breeding as well as on our lives. Here, we first review the developmental process and applications of these technologies in horticultural crop breeding. Then, we mainly describe the principles of the latest genome-editing technologies and discuss their potential applications in the genetic improvement of horticultural crops. The advantages and challenges of genome-editing technologies in horticultural crop breeding are also discussed.

Figure 1

Schematic illustration of the ZFN structure and the principle of ZFN-mediated genomic modifications. The target site of the ZFN is recognized by the “left” and “right” half monomer that each consist of a tandem array of engineered ZFPs, and each engineered ZFP can recognize a nucleotide triplet (shown in different colors). The ZFN monomer is comprised of an N-terminal domain containing a NLS (red), a recognition domain that usually comprises tandem ZFPs (in different colors) and a C-terminal function domain that comprises the Fok I endonuclease. Recognition of the target sequence by the left and right ZFPs results in dimerization of the Fok I endonuclease, which is critical for the activity of the ZFNs. DNA cleavage takes place between the two ZFP recognition sites that contain a spacer sequence that is usually 6 bp long. Induced DSB of the target DNA are repaired either by NHEJ or HDR, resulting in gene mutation around the cleavage sites. NLS, nuclear localization signal; ZFP, zinc finger proteins; DSB, double-strand breaks; NHEJ, non-homologous end joining; HDR, homology-directed repair. Mutation#x002A;, the red color box region contains nucleotide deletion, insertion or substitution. Figure modified from Gaj et al. (2013), Figure 1 and Moore et al. (2012), Figure 1.

Figure 2

The structure of TALEN and the principle of TALEN-mediated genomic modifications. The target site of TALEN is recognized by the “left” and “right” half monomer that each consist of a tandem repeat of TALE repeats. Each TALE repeat comprises a 34 amino acid (aa) unit that differs at two hypervariable aa located at the 12th and 13th position, known as RVD, which determine the recognition specificity of each repeat. The TALEN monomer consists of an N-terminal domain containing a nuclear localization signal (NLS, red), a recognition domain typically composed of tandem TALE repeats (in different colors), and a C-terminal function domain that comprises the Fok I endonuclease. Simultaneous bindings of the left and right TALE enable dimerization of the Fok I cleavage domain, resulting in DSBs of the target DNA. Induced DSBs of the target DNA are repaired either by NHEJ or HDR resulting in gene mutations that include nucleotide insertion, deletion, or substitution around the cleavage site. TALE, transcription activator-like effector; NLS, nuclear localization signal; RVD, repeat-variable di-residues; DSB, double-strand breaks; NHEJ,non-homologous end joining; HDR, homology-directed repair. Mutation*, red color box regions contain nucleotide deletion, insertion or substitution. Figure modified from Gaj et al. (2013), Figure 1 and Moore et al. (2012), Figure 1.

Figure 3

Schematic illustration of the CRISPR/Cas9 system structure and principle of CRISPR/Cas9-mediated genomic modifications. The synthetic guide RNA (sgRNA) contains a region (usually 20 bp in length) complementary to the target site on the genomic loci and stem loops that mediate the binding of the Cas9 protein. The protospacer adjacent motif (PAM, NGG) required for cleavage is indicated in red, the Cas9 protein is shown by the brown circle, and the cleavage sites located 3 bp from the PAM motif are indicated by scissors. Induced DSBs of the target DNA are repaired either by NHEJ or HDR resulting in gene mutations that include nucleotide insertion, deletion or substitution around the cleavage sites. sgRNA, synthetic guide RNA; DSB, double-strand breaks; NHEJ, non-homologous end joining; HDR, homology-directed repair. Mutation*, red color box region contains nucleotide deletion, insertion or substitution. Figure modified from Xie and Yang (2013), Figure 1.