The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato.

Q2 Agricultural and Biological Sciences
生物设计研究(英文) Pub Date : 2022-12-15 eCollection Date: 2022-01-01 DOI:10.34133/bdr.0001
Tien Van Vu, Ngan Thi Nguyen, Jihae Kim, Swati Das, Jinsu Lee, Jae-Yean Kim
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引用次数: 2

Abstract

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA (gRNA) has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs (pegRNAs) negatively affected the cleaving activity of the Cas9 nuclease. The self-complementarity between the primer binding sequences (PBSs) and spacer sequence might pose risks to the activity of the Cas9 complex. However, modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein (SlMBP21), alcobaca (SlALC), and acetolactate synthase 1 (SlALS1) loci. Our data show challenges of the PE approach in tomato, indicating that a further improvement of the PE system for successful applications is demanded, such as the use of improved expression systems for enriching active PE complexes.

Abstract Image

Abstract Image

番茄Prime编辑应用的障碍及潜在的解决思路
精确的基因组编辑是作物改良的迫切需要。最近出现的CRISPR/Cas技术在精确植物基因组工程中提供了巨大的潜在应用。将逆转录酶(RT)与Cas9内切酶和“启动”延伸引导RNA(gRNA)相结合的引物编辑(PE)方法在哺乳动物细胞和几种植物物种中显示出高频率的精确基因组修饰。然而,PE方法在双子叶植物中的应用仍然有限且效率低下。我们设计并测试了引物编辑器,用于在瞬态分析中精确编辑合成序列,并通过稳定转化在番茄中获得10个基因座的理想等位基因。我们通过靶向深度测序获得的数据也显示,在烟草和番茄系统中,PE效率都很低。对PE组分活性的进一步评估发现,RT与Cas9的融合和PE gRNA(pegRNA)的结构对Cas9核酸酶的切割活性产生了负面影响。引物结合序列(PBSs)和间隔区序列之间的自互补性可能对Cas9复合物的活性构成风险。然而,通过缩短PBS或将错配引入PBS以降低其熔融温度来修饰pegRNA序列,并不能提高MADS-box蛋白(SlMBP21)、alcobaca(SlALC)和乙酰乳酸合成酶1(SlALS1)基因座的PE效率。我们的数据显示了PE方法在番茄中的挑战,表明需要进一步改进PE系统才能成功应用,例如使用改进的表达系统来富集活性PE复合物。
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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来源期刊
CiteScore
3.90
自引率
0.00%
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0
审稿时长
12 weeks
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