An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants(1,2). Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment(3), CRISPR-Cas9 integration increases the chance of off-target modifications(4), and foreign DNA sequences cause legislative concerns about genetically modified organisms(5). Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice(6), Petunia(7), grapevine, apple(8) and potato(9), or into embryo cells by biolistic bombardment in maize(10) and wheat(11). However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes(12) and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.