Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants.

ABSTRACT

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.