Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres

Abstract

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 transformed Nicotiana benthamiana using 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.

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

“…Several studies on epigenome editing in plants have been reviewed previously [ 151 ]. Direct fusions of dCas9 with fluorescent proteins have been used for chromosome imaging ( Figure 5 C) [ 152 , 153 , 154 ].…”

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

“…Further manipulation of the sgRNA provides the possibility of CRISPR/Casmediated targeting of different kinds of enzyme activities to specific sites in the genome. The incoporation of MS2 and other kinds of aptamers into the sgRNA for sequence-specific protein binding has been used in plants for transcriptional control, live imaging of chromosomes (Khosravi et al 2020;Lowder et al 2018), as well as for base editing. Multiplexing techniques require the simultaneous expression of multiple guide RNAs.…”

CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences

“…The authors showed that telomeres are localized in the periphery of interphase nuclei. However, in comparison with FISH, the efficiency of a telomere labeling by dCas9 was 70% [129]. To improve the labeling efficiency of CRISPR/dCas9 system, various orthologues of Sp-Cas9, including St1-Cas9 and Sa-Cas9, can be recruited in combination with modified sgRNAs with an RNA aptamer MS2/PP7 insertions that bind to a fluorescent coat protein [129].…”

“…However, in comparison with FISH, the efficiency of a telomere labeling by dCas9 was 70% [129]. To improve the labeling efficiency of CRISPR/dCas9 system, various orthologues of Sp-Cas9, including St1-Cas9 and Sa-Cas9, can be recruited in combination with modified sgRNAs with an RNA aptamer MS2/PP7 insertions that bind to a fluorescent coat protein [129]. Using this method, the dynamics of telomeres and centromeres in living plant cells can be traced.…”