Sora Lee, Su Hyun Park, Yu Jeong Jeong, Soyoung Kim, Bo Ryeong Kim, Bo-Keun Ha, Chaein Na, Jiyoung Lee, Jae Cheol Jeong, Cha Young Kim
{"title":"优化 CRISPR/Cas9 核糖核蛋白向卷心菜原生质体的输送,实现高效的无 DNA 基因编辑","authors":"Sora Lee, Su Hyun Park, Yu Jeong Jeong, Soyoung Kim, Bo Ryeong Kim, Bo-Keun Ha, Chaein Na, Jiyoung Lee, Jae Cheol Jeong, Cha Young Kim","doi":"10.1007/s11816-024-00901-9","DOIUrl":null,"url":null,"abstract":"<p>The CRISPR/Cas9-based gene editing system for the direct delivery of pre-assembled Cas9 ribonucleoproteins (RNPs), consisting of a Cas9 nuclease and a single guide RNA (sgRNA), into plant protoplasts enables DNA-free gene editing without introducing foreign gene into plants. Here, we described the optimization of CRISPR/Cas9 RNPs delivery into cabbage protoplasts for efficient DNA-free gene editing. We determined the insertion and deletion (indel) frequency of <i>BoMYBL2-1</i>, a negative regulatory gene for anthocyanin biosynthesis in cabbage (<i>Brassica oleracea</i> var. <i>capitata</i>). We optimized the molar ratio of Cas9 to sgRNA and the incubation time of RNP–protoplast transfection to enhance the indel frequency under various conditions. Based on the <i>BoMYBL2-1</i> nucleotide sequences, we designed nine sgRNAs to target <i>BoMYBL2-1</i>. Our <i>in vitro</i> digestion assay showed that all sgRNAs were able to cleave the targeted fragment. When the sgRNA and Cas9 proteins were subsequently transfected into protoplasts isolated from cabbage cotyledons, the deep sequencing results showed that the indel frequency of sgRNAs in <i>BoMYBL2-1</i> was the highest (7.4%) with sgRNA3. We compared various molar ratios of Cas9 and sgRNA and incubation times of RNP–protoplast transfection to optimize transfection and ensure high indel frequency. The highest frequency was observed when the Cas9:sgRNA ratio was 1:10. Furthermore, when the incubation time for RNP–protoplast transfection was 1 min and 3 min, the indel frequency was higher than 25%. Altogether, these results provide valuable information on the optimized conditions for high-efficiency gene editing using CRISPR/Cas9 RNP delivery into cabbage protoplasts.</p>","PeriodicalId":20216,"journal":{"name":"Plant Biotechnology Reports","volume":null,"pages":null},"PeriodicalIF":1.7000,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Optimization of CRISPR/Cas9 ribonucleoprotein delivery into cabbage protoplasts for efficient DNA-free gene editing\",\"authors\":\"Sora Lee, Su Hyun Park, Yu Jeong Jeong, Soyoung Kim, Bo Ryeong Kim, Bo-Keun Ha, Chaein Na, Jiyoung Lee, Jae Cheol Jeong, Cha Young Kim\",\"doi\":\"10.1007/s11816-024-00901-9\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>The CRISPR/Cas9-based gene editing system for the direct delivery of pre-assembled Cas9 ribonucleoproteins (RNPs), consisting of a Cas9 nuclease and a single guide RNA (sgRNA), into plant protoplasts enables DNA-free gene editing without introducing foreign gene into plants. Here, we described the optimization of CRISPR/Cas9 RNPs delivery into cabbage protoplasts for efficient DNA-free gene editing. We determined the insertion and deletion (indel) frequency of <i>BoMYBL2-1</i>, a negative regulatory gene for anthocyanin biosynthesis in cabbage (<i>Brassica oleracea</i> var. <i>capitata</i>). We optimized the molar ratio of Cas9 to sgRNA and the incubation time of RNP–protoplast transfection to enhance the indel frequency under various conditions. Based on the <i>BoMYBL2-1</i> nucleotide sequences, we designed nine sgRNAs to target <i>BoMYBL2-1</i>. Our <i>in vitro</i> digestion assay showed that all sgRNAs were able to cleave the targeted fragment. When the sgRNA and Cas9 proteins were subsequently transfected into protoplasts isolated from cabbage cotyledons, the deep sequencing results showed that the indel frequency of sgRNAs in <i>BoMYBL2-1</i> was the highest (7.4%) with sgRNA3. We compared various molar ratios of Cas9 and sgRNA and incubation times of RNP–protoplast transfection to optimize transfection and ensure high indel frequency. The highest frequency was observed when the Cas9:sgRNA ratio was 1:10. Furthermore, when the incubation time for RNP–protoplast transfection was 1 min and 3 min, the indel frequency was higher than 25%. Altogether, these results provide valuable information on the optimized conditions for high-efficiency gene editing using CRISPR/Cas9 RNP delivery into cabbage protoplasts.</p>\",\"PeriodicalId\":20216,\"journal\":{\"name\":\"Plant Biotechnology Reports\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.7000,\"publicationDate\":\"2024-04-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Biotechnology Reports\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1007/s11816-024-00901-9\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Biotechnology Reports","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s11816-024-00901-9","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Optimization of CRISPR/Cas9 ribonucleoprotein delivery into cabbage protoplasts for efficient DNA-free gene editing
The CRISPR/Cas9-based gene editing system for the direct delivery of pre-assembled Cas9 ribonucleoproteins (RNPs), consisting of a Cas9 nuclease and a single guide RNA (sgRNA), into plant protoplasts enables DNA-free gene editing without introducing foreign gene into plants. Here, we described the optimization of CRISPR/Cas9 RNPs delivery into cabbage protoplasts for efficient DNA-free gene editing. We determined the insertion and deletion (indel) frequency of BoMYBL2-1, a negative regulatory gene for anthocyanin biosynthesis in cabbage (Brassica oleracea var. capitata). We optimized the molar ratio of Cas9 to sgRNA and the incubation time of RNP–protoplast transfection to enhance the indel frequency under various conditions. Based on the BoMYBL2-1 nucleotide sequences, we designed nine sgRNAs to target BoMYBL2-1. Our in vitro digestion assay showed that all sgRNAs were able to cleave the targeted fragment. When the sgRNA and Cas9 proteins were subsequently transfected into protoplasts isolated from cabbage cotyledons, the deep sequencing results showed that the indel frequency of sgRNAs in BoMYBL2-1 was the highest (7.4%) with sgRNA3. We compared various molar ratios of Cas9 and sgRNA and incubation times of RNP–protoplast transfection to optimize transfection and ensure high indel frequency. The highest frequency was observed when the Cas9:sgRNA ratio was 1:10. Furthermore, when the incubation time for RNP–protoplast transfection was 1 min and 3 min, the indel frequency was higher than 25%. Altogether, these results provide valuable information on the optimized conditions for high-efficiency gene editing using CRISPR/Cas9 RNP delivery into cabbage protoplasts.
期刊介绍:
Plant Biotechnology Reports publishes original, peer-reviewed articles dealing with all aspects of fundamental and applied research in the field of plant biotechnology, which includes molecular biology, genetics, biochemistry, cell and tissue culture, production of secondary metabolites, metabolic engineering, genomics, proteomics, and metabolomics. Plant Biotechnology Reports emphasizes studies on plants indigenous to the Asia-Pacific region and studies related to commercialization of plant biotechnology. Plant Biotechnology Reports does not exclude studies on lower plants including algae and cyanobacteria if studies are carried out within the aspects described above.