The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding

Abstract

Genome editing with engineered nucleases enabling site-directed sequence modifications bears a great potential for advanced plant breeding and crop protection. Remarkably, the RNA-guided endonuclease technology (RGEN) based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) is an extremely powerful and easy tool that revolutionizes both basic research and plant breeding. Here, we review the major technical advances and recent applications of the CRISPR-Cas9 system for manipulation of model and crop plant genomes. We also discuss the future prospects of this technology in molecular plant breeding.

Figure 1

Overview of CRIPR-Cas9 technology for plant genome editing. (A) The most widely used engineered CRISPR-Cas9 system in plants utilizes a plant-codon-optimized Cas9 protein and (could be more than one) single-guide RNA (sgRNA). Optionally, the gene targeting system with geminivirus replicons includes an additional donor DNA template. (B) In plant cells, sgRNA associated with Cas9 nuclease mediates cleavage of target DNA sites that are complementary to the sgRNA and locate next to a PAM sequence. (C) Cas9-induced double-strand DNA breaks (DSBs) can be repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. (D) Imprecise NHEJ-mediated repair can generate insertion and/or deletion mutations with variable length at the site of the DSB. These InDels can cause out-of-frame mutations in the coding sequences of the target genes, resulting in gene knockout. (E) In the presence of donor DNA, NHEJ can insert the donor DNA into the site of the DSB together with possibly additional InDel mutations. HR-driven repair can produce precise modifications, including point mutations (F) or insertions from double-/single-strand DNAs as donor templates (G).