开发用于水稻核心启动子编辑的 CRISPR/FrCas9 系统

IF 4.6 4区 农林科学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Hui Wang, Jian Ding, Jingyan Zhu, Xiaoshuang Liu, Rongfang Xu, Ruiying Qin, Dongfang Gu, Min Li, Pengcheng Wei, Juan Li
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引用次数: 0

摘要

基因核心启动子区域的微小突变可能会导致表达强度发生重大变化。然而,靶向核心启动子中富含 TA 的序列可能会给 SpCas9 和其他富含 G 的 PAM 兼容 Cas9 等 Cas9 变体带来挑战。在这项研究中,我们为植物基因组编辑设计了一种独特的 FrCas9 系统,该系统来源于啮齿动物粪杆菌。我们的研究结果表明,当使用 TATA 序列作为 PAM 时,该系统在水稻中是有效的。此外,FrCas9 对所有 16 种可能的 NNTA PAM 都具有活性,在胼胝体中的效率高达 35.3%,并在 31.3% 的 T0 转基因植株中产生同源或双拷贝突变。一项研究水稻 WX 核心启动子编辑的概念验证实验证实,FrCas9 诱导的突变可改变基因表达和直链淀粉含量。通过 FrCas9 介导的双向编辑,以单个 palindromic TATA 序列作为 PAM,产生了多重突变和缺失。此外,我们还开发了源于 FrCas9 的碱基编辑器,能够在植物的 A-T 和 G-C 对之间进行可编程转换。这项研究强调了用于植物核心启动子编辑的多功能 FrCas9 工具集,为基因表达的微调和新种质的创造提供了巨大的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Developing a CRISPR/FrCas9 system for core promoter editing in rice

Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T0 transgenic plants. A proof-of-concept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms.

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CiteScore
7.70
自引率
2.80%
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