Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Abstract

Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

Keywords: CRISPR/Cas9; PAM; SgRNA; gene editing; plant molecular breeding.

Figure 1

Principle of CRISPR/Cas9 gene-editing technology and targeting genome: (a) Mechanism of CRISPR/Cas9-mediated DNA repair; (b) CRISPR/Cas9 targets different plant genomes. CRISPR/Cas9 targeting of the target genome first produces a double-stranded DNA break, followed by two DNA repair mechanisms, non-homologous end-joining, and homologous recombination, in which editing occurs during the repair process. Non-homologous end-joining often produces deletions and insertions of target sequences, and homologous recombination produces exchanges of target sequence fragments or base substitutions. The figure mentions the names of the genes recently targeted by CRISPR/Cas9 in different major crops.