Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres
Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres
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DOI：
10.3389/fpls.2020.01254
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发表时间：
2020-08-20
影响因子：
5.6
通讯作者：
Houben, Andreas
中科院分区：
文献类型：
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作者：
Khosravi, Solmaz;Schindele, Patrick;Houben, Andreas
发展实时成像技术以提供染色质在活细胞中如何组织的信息，对于破译生物过程的调节至关重要。在这里，我们展示了一种基于CRISPR/CAS9的实时成像技术的改进。在这种方法中，sgRNA支架与包括MS2和PP7在内的RNA适配子融合。当死亡的Cas9(DCas9)与嵌合的sgRNA共表达时，标记为MS2和PP7适配子的荧光外壳蛋白(tdMCP-FP和tdPCP-FP)被招募到靶序列中。与以前使用dCas9：GFP进行的工作相比，我们发现使用基于适体的CRISPR成像构建体瞬时转化本底烟草的端粒标记质量得到了改善。标记受适配子拷贝数的影响较小，受启动子类型的影响较小。同样的结构不适用于稳定转化的植物和根中重复序列的标记。RNP复合体与其靶DNA的持续相互作用可能会干扰细胞过程。
Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformedNicotiana benthamianausing aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes.