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

IF 1.7 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Plant Biotechnology Reports Pub Date : 2024-04-10 DOI:10.1007/s11816-024-00901-9
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
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引用次数: 0

摘要

基于 CRISPR/Cas9 的基因编辑系统可将预先组装好的 Cas9 核糖核蛋白(RNPs)(由 Cas9 核酸酶和单导 RNA(sgRNA)组成)直接输送到植物原生质体中,从而实现无 DNA 基因编辑,且不会将外来基因引入植物。在此,我们介绍了如何优化将 CRISPR/Cas9 RNPs 导入卷心菜原生质体以实现高效的无 DNA 基因编辑。我们测定了卷心菜(Brassica oleracea var. capitata)花青素生物合成负调控基因 BoMYBL2-1 的插入和缺失(indel)频率。我们优化了 Cas9 与 sgRNA 的摩尔比以及 RNP-表皮转染的孵育时间,以提高各种条件下的吲哚频率。根据BoMYBL2-1的核苷酸序列,我们设计了9种靶向BoMYBL2-1的sgRNA。体外消化实验表明,所有的sgRNA都能裂解靶向片段。我们比较了Cas9和sgRNA的不同摩尔比以及RNP-原生质体转染的孵育时间,以优化转染并确保高吲哚频率。当 Cas9 与 sgRNA 的比例为 1:10 时,我们观察到了最高的吲哚频率。此外,当 RNP-原核细胞转染的孵育时间为 1 分钟和 3 分钟时,吲哚频率高于 25%。总之,这些结果为利用 CRISPR/Cas9 RNP 向卷心菜原生质体传递高效基因编辑的优化条件提供了有价值的信息。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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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.

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来源期刊
Plant Biotechnology Reports
Plant Biotechnology Reports 生物-生物工程与应用微生物
CiteScore
4.10
自引率
4.20%
发文量
72
审稿时长
>12 weeks
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.
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