CRISPR/Cas9-Based Genome Editing Using Rice Zygotes

Genome-editing technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Its application has been hindered in many cases, however, due to technical hurdles, such as the low rate of macromolecule delivery into plant cells and tissues or difficulties in plant transformation and regeneration. Here, a protocol for CRISPR/Cas9-based genome editing using rice zygotes is described. The genome-editing system is constructed via polyethylene glycol/calcium-mediated transfection with CRISPR/Cas9 components in rice zygotes, which are produced by in vitro fertilization of isolated rice gametes. Plasmid DNA harboring a CRISPR/Cas9 expression cassette or preassembled Cas9 protein–guide RNA ribonucleoproteins is transfected into zygotes, resulting in the regeneration of plants with a high frequency of the targeted mutation, which is either mono-allelic or bi-allelic, in the range of about 4% to 64%. Application of the present method has the potential to advance the molecular breeding of other crop species as well as rice. © 2020 Wiley Periodicals LLC.