Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering

Abstract

Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.

Keywords: CRISPR/Cas9; CRISPR/dCas9; Null segregant; Plant genome engineering.

Fig. 1

Recent applications of base editing (a) and epigenetic editing (b) technologies in plants. a Co-editing of two genes (TaALS and TaACCase) by using dCas9 fused with CBE [27]. The approach produced nicosulfuron-resistant wheat plants with mutations in the two genes but without transgenes. b Targeted demethylation of the FWA promoter by using dCas9-Suntag-hTET1cd in Arabidopsis [28]. The approach resulted in the activation of FWA gene expression. Demethylation and late-flowering phenotypes were inherited by T2 generation without transgenes

Fig. 2

Generation of null segregants in plants by CRISPR/Cas9 technology. Representative methods for the production of null segregants are shown: isolation of null segregants by Mendelian segregation (a); programmed self-elimination of transgenic plants (b); transient expression of CRISPR/Cas9 (c); and ribonucleoprotein-mediated genome editing (d). Please refer to the text for detailed explanations. “M” and “T”, plants with mutation and transgene insertion, respectively