Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases.

IF 17.1 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Molecular Plant Pub Date : 2024-05-06 Epub Date: 2024-03-22 DOI:10.1016/j.molp.2024.03.013
Tom Schreiber, Anja Prange, Petra Schäfer, Thomas Iwen, Ramona Grützner, Sylvestre Marillonnet, Aurélie Lepage, Marie Javelle, Wyatt Paul, Alain Tissier
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Abstract

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double-strand breaks, making it challenging to generate knock-in events. In this study, we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrated stable and heritable knock-ins in wheat in 1% of the primary transformants. Taken together, our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

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通过工程化的 CRISPR/Cas 内切酶,在植物中高效无痕地敲入几千个碱基。
在植物和哺乳动物中,非同源末端连接是修复 DNA 双股断裂的主要途径,因此产生基因敲入事件具有挑战性。我们从疱疹病毒和噬菌体 T7 家族中发现了两组外切核酸酶,当它们与 Cas9/Cas12a 融合后,在烟草花叶病毒为基础的本根烟草中进行瞬时实验时,HDR 频率最多可增加 38 倍。我们在瞬时转化和稳定转化系统中都实现了几千碱基 DNA 的精确无痕插入。在拟南芥中,将 Cas9 与疱疹病毒家族的外切酶融合可使第一代转化子中的基因敲入频率提高 10 倍。此外,我们还证明了小麦中 1%的初代转化子具有稳定的遗传性基因敲入。我们的研究结果为植物中遗传性基因敲入和基因替换事件的常规生产开辟了前景。
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来源期刊
Molecular Plant
Molecular Plant 植物科学-生化与分子生物学
CiteScore
37.60
自引率
2.20%
发文量
1784
审稿时长
1 months
期刊介绍: Molecular Plant is dedicated to serving the plant science community by publishing novel and exciting findings with high significance in plant biology. The journal focuses broadly on cellular biology, physiology, biochemistry, molecular biology, genetics, development, plant-microbe interaction, genomics, bioinformatics, and molecular evolution. Molecular Plant publishes original research articles, reviews, Correspondence, and Spotlights on the most important developments in plant biology.
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