开发用于水稻核心启动子编辑的 CRISPR / FrCas9 系统。Developing a CRISPR/FrCas9 system for core promoter editing in rice.-医云文献数字医云科研云海量医学决策数据服务

Abstract：

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.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00157-5.

摘要：

基因核心启动子区中的小突变可导致表达强度的实质性变化。然而,靶向核心启动子的富含TA的序列可能对Cas9变体(诸如SpCas9和其他富含G的PAM相容性Cas9s)构成挑战。在这项研究中,我们设计了一个独特的FrCas9系统，用于植物基因组编辑。我们的发现表明，当TATA序列用作PAM时，该系统在水稻中是有效的。此外,FrCas9证明了对所有16种可能的NNTAPAM的活性，在愈伤组织中达到高达35.3％的效率，并在31.3％的T0转基因植物中产生纯合或双等位基因突变。用于检查水稻WX核心启动子编辑的概念验证实验证实，FrCas9诱导的突变可以改变基因表达和直链淀粉含量。双向编辑产生多重突变和缺失，由FrCas9介导，使用单个回文TATA序列作为PAM。此外，我们开发了FrCas9衍生的基础编辑器，能够在植物中的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。
■在线版本包含补充材料，可在10.1007/s42994-024-00157-5获得。