Development of a CRISPR-imaging toolset for imaging of DNA and RNA in structurally preserved chromatin suitable for super-resolution microscopy and electron microscopy and its application to study the substructure of holo- and monocentromeres

• Applicant: Privatdozent Dr. Andreas Houben
• Subject Area: Plant Genetics and Genomics ; Structural Biology
• Term: from 2021 to 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 462333861

• Final Report Year: 2025

Final Report Abstract

The temporal and spatial organization of chromosomes and nuclei is increasingly recognized as being important for regulating functions such as gene expression, DNA replication and repair, as well as proper segregation of the genetic material during cell division. Within the framework of the DFG-funded project, we extended the CRISPR-imaging toolset with newly developed fluorescent and non-fluorescent-based methods for labelling defined genomic sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR-imaging eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Using an ALFA-tagged dCas9 protein assembled with a target sequence-specific gRNA, we demonstrate specific labelling of target sequences with a fluorescence-labeled ALFA-nanobody. dCas9 possessing multiple copies of the ALFA-tag, in combination with a minibody, resulted in enhanced target-specific fluorescence signals compared to previous approaches. The application of the Cas9-ALFA-tag system in combination with FluoroNanogold suitable is possible. However, the detection of FluoroNanogold-based signals by for electron microscopic was impossible because the transfer of CRISPR-labelled specimens to electron microscopy grids failed. Furthermore, we successfully linked the highly sensitive Tyramide Signal Amplification (TSA) method with CRISPR-FISH. To develop a nonfluorescent-based imaging method, we combined chromogenic signal detection using alkaline phosphatase or peroxidase enzymes with CRISPR imaging (CRISPR-CID). This advancement makes DNA labelling techniques accessible to researchers and schools in resource-limited settings, because for signal detection a standard ligh microscope is sufficient. The application of quantum dots and the detection of RNA and single copy sequences in combination with CRISPR-imaging was not successful. In addition, CRISPR-based live imaging was further developed. The application of an intronized dCas9 variant enhanced the signal intensity and, for the first time, enabled the live imaging of centromeres and rDNA repeats in transiently transformed Nicotiana benthamiana.

Publications

• CRISPR-FISH: A CRISPR/Cas9-Based In Situ Labeling Method. Methods in Molecular Biology, 315-335. Springer US.
  Potlapalli, Bhanu Prakash; Ishii, Takayoshi; Nagaki, Kiyotaka; Somasundaram, Saravanakumar & Houben, Andreas
  
• Application of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit v1. ZappyLab, Inc..
  Prakash Potlapalli, Bhanu; Fuchs, Joerg; Rutten, Twan; Meister, Armin & Houben, Andreas
  
• Genomische Sequenzen sichtbar machen. Biologie in unserer Zeit 54:37 - 40
  Houben A., Potlapalli B.P. & Khosravi S.
  
• The potential of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit. Journal of Experimental Botany, 75(20), 6244-6257.
  Potlapalli, Bhanu Prakash; Fuchs, Jörg; Rutten, Twan; Meister, Armin & Houben, Andreas
  
• CRISPR-CISH: an in situ chromogenic DNA repeat detection system for research and life science education. Chromosome Research, 33(1).
  Potlapalli, Bhanu Prakash; Dassau, Fabian; Fuchs, Jörg; Sushmoy, Deboprio Roy & Houben, Andreas