Abstract

The peculiarities in the creation of genetic engineering tools for the knock-in variant of genome editing are considered in detail on the example of gfp gene delivery to two target regions (nucleolus organizer region and the region of one of histone H3 genes) of the Arabidopsis thaliana (L.) Heynh. genome using different methods of delivering exogenous DNA (agrobacterium-mediated transformation, bio-ballistics using different vectors and RNP complexes). Differences in the approaches to the creation of donor constructions and Cas9 tools depending on the selected method of delivery are considered. The selected target regions are of interest for further biotechnological studies on the creation of recombinant protein bioproducer lines since they refer to “housekeeping” gene regions and are characterized by a high transcriptional activity. It was established that the selected regions are not equivalent to each other as targets for incorporation of exogenous DNA. A complex compartmentalization of nucleolus, as well as a unique mechanism of “neutralization” of double-strand breaks, acts as barriers preventing the delivery of genetic engineering tools to this region. The second region of “housekeeping” genes (histone Н3.3 gene region) seems accessible and can be used for the knock-in variant of genome editing.

中文翻译：

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拟南芥细胞培养基因组编辑基因敲入变体的基因工程构建创造中的特殊性

摘要

在将gfp基因传递至拟南芥的两个靶标区域（核仁组织者区域和组蛋白H3基因之一的区域）的示例中，详细考虑了基因组编辑敲入变体的基因工程工具的创建中的特殊性。拟南芥（L.）Heynh。使用不同的方式递送外源DNA的基因组（农杆菌介导的转化，使用不同载体和RNP复合物的生物弹道学）。考虑了根据所选的交付方式创建供体结构和Cas9工具的方法的差异。所选择的靶区域对于重组蛋白生物生产者系的产生的进一步的生物技术研究是有意义的，因为它们指的是“持家”基因区域，并且具有高转录活性。已经确定，选择的区域彼此不同，作为掺入外源DNA的靶标。核仁的复杂区室化以及双链断裂“中和”的独特机制，阻碍了基因工程工具向该地区的运输。“管家”基因的第二个区域（组蛋白Н3.3基因区域）似乎可访问，可用于基因组编辑的敲入变体。