Researchers from Tokyo Metropolitan University presented a new protocol for CRISPR-Cas9ā?based Genome-editing using rice zygotes. This addresses some technical hurdles faced in the application of gene-editing technology. The description of the technique is published in Current Protocols.
Genome-editing technology for targeted mutagenesis of plants using programmable nucleases has been extensively used for next-generation plant breeding. Just like any new technology, there are some technical challenges in the application, such as low rate of macromolecule delivery into plant cells and tissues or difficulties in plant transformation and regeneration. Thus, Erika Roda and Takashi Okamoto developed a new protocol using rice zygotes. The genome-editing system is developed via polyethylene glycol/calciumā?mediated transfection with CRISPR-Cas9 components in rice zygotes, which are produced by in vitro fertilization of isolated rice gametes. The plasmid DNA harboring a CRISPR-Cas9 expression cassette or preassembled Cas9 proteinā??guide RNA ribonucleoproteins are transfected into zygotes, leading to the regeneration of plants with a high frequency of the targeted mutation.
This new protocol has the potential to improve the molecular breeding methods of rice and other crops.