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Porcine zona pellucida\u2010free zygotes and embryos were incubated for 5\u00a0h with CRISPR\u2010associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10\u00a0h post\u2010in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29\u2009h post\u2010in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were &lt;34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry\u2010based technology needs further improvement, especially regarding editing efficiency and embryo development.<\/jats:p>","DOI":"10.1111\/asj.13878","type":"journal-article","created":{"date-parts":[[2023,10,12]],"date-time":"2023-10-12T05:11:25Z","timestamp":1697087485000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Comparison of chemically mediated CRISPR\/Cas9 gene editing systems using different nonviral vectors in porcine embryos"],"prefix":"10.1111","volume":"94","author":[{"given":"Qingyi","family":"Lin","sequence":"first","affiliation":[{"name":"Bio\u2010Innovation Research Center Tokushima University  Tokushima Japan"},{"name":"Faculty of Bioscience and Bioindustry Tokushima University  Tokushima 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