The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology

Abstract

Genome editing technologies have progressed rapidly and become one of the most important genetic tools in the implementation of pathogen resistance in plants. Recent years have witnessed the emergence of site directed modification methods using meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Recently, CRISPR/Cas9 has largely overtaken the other genome editing technologies due to the fact that it is easier to design and implement, has a higher success rate, and is more versatile and less expensive. This review focuses on the recent advances in plant protection using CRISPR/Cas9 technology in model plants and crops in response to viral, fungal and bacterial diseases. As regards the achievement of viral disease resistance, the main strategies employed in model species such as Arabidopsis and Nicotiana benthamiana, which include the integration of CRISPR-encoding sequences that target and interfere with the viral genome and the induction of a CRISPR-mediated targeted mutation in the host plant genome, will be discussed. Furthermore, as regards fungal and bacterial disease resistance, the strategies based on CRISPR/Cas9 targeted modification of susceptibility genes in crop species such as rice, tomato, wheat, and citrus will be reviewed. After spending years deciphering and reading genomes, researchers are now editing and rewriting them to develop crop plants resistant to specific pests and pathogens.

Keywords: CRISPR/Cas9; bacteria; crop improvement; disease resistance; fungus; genome editing; virus.

FIGURE 1

Illustrative diagram of Cas9 and Cpf1 activities. The target specificity is given by the 17-20 nt located at the 5′ end of the sgRNA sequence. (A) Primary transcript and gRNA-nuclease (Cas9 or Cpf1) complex formation. The catalytic domains are RUV-C (light blue) and HNH (yellow) for Cas9 and RUV-C for Cpf1. The Cas9 is colored in light blue and the Cpf1 in dark blue; in black is represented the gRNA for gene targeting. (B) Gene target activity. Cas9 has 5′-NGG-3′ PAM sequence (blue bars) and Cpf1 has 5′-TTTV-3′ PAM sequence (green bars). (C) DNA ends after nuclease activity. Cas9 lead to blunt-end and Cpf1 to sticky-ends.

FIGURE 2

Illustrative diagram of polycistronic tRNA-gRNA (PTG) gene construct and targeting activity for Cas9. PTG is composed of t-RNA-gRNA repeats and is upregulated by ZmU6 promoter or TaU3 promoter according the experimental design as different terminator region (T) are adopted. (A) PTG primary transcript. Endogenous endonuclease cuts the tRNA ends and let each tRNA-gRNA targeting the corresponding gene sequence. (B) In PTG system more sequence targets are available (n° gene targets) and the different gRNA are represented in different colors (orange, pink, and green).