High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice
High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice
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DOI：
10.1038/s41598-019-55681-y
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发表时间：
2019-12-27
影响因子：
4.6
通讯作者：
Wang, Kan
中科院分区：
文献类型：
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作者：
Banakar, Raviraj;Eggenberger, Alan L.;Wang, Kan
将CRISPR试剂作为核糖核蛋白（RNP）复合物递送到细胞中的一个重要优势是能够编辑基因而无需将试剂整合到基因组中。细胞中RNP分子的瞬时存在可以减少不期望的脱靶效应。将RNP递送到植物细胞中的一种方法是使用生物射弹枪。为了在RNP递送期间促进转化细胞的选择，携带选择性标记基因的质粒可以与RNP共递送以富集转化/编辑的细胞。在这项工作中，我们比较了在水稻中使用三种不同的交付平台的靶向诱变：基因枪RNP/DNA共交付;基因枪DNA交付;和农杆菌介导的交付。所有三个平台都成功地在靶位点产生了所需的突变。然而，我们观察到高频率（超过14%）的随机质粒或染色体DNA片段插入在靶位点的转基因事件产生的两个生物射弹传递平台。与此相反，在由农杆菌介导的方法产生的转基因事件中没有观察到随机DNA片段的整合。这些数据揭示了在选择植物中基因组编辑试剂递送方法时必须考虑的重要见解，并强调了采用适当的分子筛选方法来检测基因组工程后的非预期改变的重要性。
An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.