多重CRISPR/Cas9策略在消除转基因植物选择标记中的应用

IF 4.4 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Frontiers in genome editing Pub Date : 2025-09-03 eCollection Date: 2025-01-01 DOI:10.3389/fgeed.2025.1633104
Mohammed Rafi, Mohamed ElSiddig, Maitha Aldarmaki, Mariam Al Nuaimi, Suja George, Khaled M A Amiri
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引用次数: 0

摘要

选择标记基因(smg)是鉴定转基因植物的关键,但也引起了对生物安全性、法规遵从性和公众接受度的担忧。在本研究中,我们使用基于CRISPR/ cas9的策略从转基因烟草植株中去除SMG。将携带DsRED (SMG)和氨基糖苷磷酸转移酶(感兴趣的基因,GOI)的植物叶片用含有四个grna的CRISPR载体重新转化,这些grna被设计为针对SMG盒的两侧区域。大约20%的再生芽表现出红色荧光的缺失,PCR和测序分析证实,其中约一半的再生芽携带较小的扩增子,这表明SMG的成功切除效率约为10%。突变分析进一步显示,除了SMG盒缺失外,gRNA靶点上还存在小的索引。实时荧光定量PCR (qPCR)分析证实smg缺失系中DsRED不表达,Cas9和GOI仍有活性表达。无smg的植株生长、开花和制种正常,表明CRISPR标记切除对植株发育和育性没有不利影响。此外,在T1代通过分离获得了不含cas9和无标记的转基因植株。该方法适用于多种转基因植物品种,为产生无标记转基因作物提供了切实可行的解决方案,从而提高了转基因作物的接受度和商业化程度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Application of a multiplex CRISPR/Cas9 strategy for elimination of selection markers from transgenic plants.

Application of a multiplex CRISPR/Cas9 strategy for elimination of selection markers from transgenic plants.

Application of a multiplex CRISPR/Cas9 strategy for elimination of selection markers from transgenic plants.

Application of a multiplex CRISPR/Cas9 strategy for elimination of selection markers from transgenic plants.

Selectable marker genes (SMGs) are essential for identifying transgenic plants but raise concerns regarding biosafety, regulatory compliance, and public acceptance. In this study, we used a CRISPR/Cas9-based strategy to eliminate the SMG from transgenic tobacco plants. Leaf discs from plants carrying DsRED (SMG) and aminoglycoside phosphotransferase (gene of interest, GOI) were re-transformed with a CRISPR vector containing four gRNAs designed to target both flanking regions of the SMG cassette. Approximately 20% of the regenerated shoots exhibited loss of red fluorescence, and PCR and sequencing analyses confirmed that about half of these carried a smaller amplicon, indicating a successful SMG excision efficiency of around 10%. Mutation analysis further revealed the presence of small indels at gRNA target sites, in addition to the deletion of SMG cassette. Quantitative real-time PCR (qPCR) analysis confirmed the absence of DsRED expression in SMG-deleted lines, while the Cas9 and GOI remained actively expressed. The SMG-free plants displayed normal growth, flowering, and seed production, indicating CRISPR marker excision had no adverse effects on plant development and fertility. In addition, Cas9-free, marker-free transgenic plants were recovered through segregation in T1 generation. This approach is adaptable to various transgenic plant species and provides a practical solution for generating marker-free transgenic crops, thereby enhancing their acceptance and commercialization.

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