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Development of a CRISPR-FISH toolset for imaging of genomic loci in living plants and its application to study the centromere structure and the process of chromosome condensation in living cells

Subject Area Plant Genetics and Genomics
Term from 2017 to 2021
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 358939560
 
Final Report Year 2020

Final Report Abstract

To establish a CRISPR/dCas9-based live cell imaging for plants, two catalytically inactive Cas9 orthologues of Streptococcus pyogenes (Sp-dCas9) and Staphylococcus aureus (SadCas9) were applied. Multiple copies of fluorescence proteins, either eGFP or mRuby2, were fused to the C-terminal end of each dCas9 variant. A robust visualization of telomere repeats of transiently transformed Nicotiana benthamiana revealed dynamic telomere movements of up to 2 µm within 30 minutes during interphase. Furthermore, CRISPR/dCas9-imaging can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we paved the way for imaging of multiple genomic loci in live plants cells. In order to improve the intensity of the reporter construct the sgRNA scaffold was fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the aptamer-binding proteins fused to fluorescent protein (MCP-FP and PCP-FP) are recruited to the targeted sequence. Compared to dCas9:GFP, the quality of telomere labelling was improved in transiently transformed N. benthamiana. Labelling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labelling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. A novel tool was developed to visualize defined genomic sequences in fixed plant and animal nuclei and chromosomes based on a two-part guide RNA recombinant Cas9 endonuclease complex. In contrast to classical FISH, RGEN-ISL (RNA-guided endonuclease – in situ labelling) does not require DNA denaturation and permits a better structural chromatin preservation. The application of differentially labelled tracrRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry, FISH and DNA replication studies. Real-time visualization of the CRISPR/Cas9-mediated DNA labelling process, revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to revolutionize the field of chromosome biology.

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