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

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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Abstract

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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多重CRISPR/Cas9策略在消除转基因植物选择标记中的应用
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。
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CiteScore
7.00
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0.00%
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审稿时长
13 weeks
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