用引物从pCas9-NAT质粒中扩增出Nourseothricin抗生素标记区以外的片段。通过引物从pRS42H-HXK2质粒中扩增出潮霉素B抗性标记。用Gibson Assembly®克隆试剂盒组装这两个PCR片段，构建pCas-Hig质粒。通过供体GZF3-F和供体-GZF3-R引物构建GZF3基因缺失的供体DNA。每个60 bp的正向和反向引物都包含一个互补的30 bp同源区。将pCas9-Hyg、pRS42K-gRNA-CAR1和CAR1供体DNA转化到酿酒酵母GRL6中，以缺失CAR1基因。
参考文献：Jung JY, Kang MJ, Hwang HS, Baek KR, Seo SO. Reduction of Ethyl Carbamate in an Alcoholic Beverage by CRISPR/Cas9-Based Genome Editing of the Wild Yeast. Foods. 2022 Dec 25;12(1):102.
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用引物从pCas9-NAT质粒中扩增出Nourseothricin抗生素标记区以外的片段。通过引物从pRS42H-HXK2质粒中扩增出潮霉素B抗性标记。用Gibson Assembly®克隆试剂盒组装这两个PCR片段，构建pCas-Hig质粒。通过供体GZF3-F和供体-GZF3-R引物构建GZF3基因缺失的供体DNA。每个60 bp的正向和反向引物都包含一个互补的30 bp同源区。将pCas9-Hyg、pRS42K-gRNA-CAR1和CAR1供体DNA转化到酿酒酵母GRL6中，以缺失CAR1基因。
参考文献：Jung JY, Kang MJ, Hwang HS, Baek KR, Seo SO. Reduction of Ethyl Carbamate in an Alcoholic Beverage by CRISPR/Cas9-Based Genome Editing of the Wild Yeast. Foods. 2022 Dec 25;12(1):102.
