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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\u2010based genome editing using rice zygotes is described. The genome\u2010editing system is constructed via polyethylene glycol\/calcium\u2010mediated transfection with CRISPR\/Cas9 components in rice zygotes, which are produced by <jats:italic>in vitro<\/jats:italic> fertilization of isolated rice gametes. Plasmid DNA harboring a CRISPR\/Cas9 expression cassette or preassembled Cas9 protein\u2013guide RNA ribonucleoproteins is transfected into zygotes, resulting in the regeneration of plants with a high frequency of the targeted mutation, which is either mono\u2010allelic or bi\u2010allelic, 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. \u00a9 2020 Wiley Periodicals LLC.<\/jats:p>","DOI":"10.1002\/cppb.20111","type":"journal-article","created":{"date-parts":[[2020,6,9]],"date-time":"2020-06-09T13:01:47Z","timestamp":1591707707000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["CRISPR\/Cas9\u2010Based Genome Editing Using Rice Zygotes"],"prefix":"10.1002","volume":"5","author":[{"given":"Erika","family":"Toda","sequence":"first","affiliation":[{"name":"Department of Biological Sciences Tokyo Metropolitan University  Hachioji Tokyo Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takashi","family":"Okamoto","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Tokyo Metropolitan University  Hachioji Tokyo 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