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<p>In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.</p>

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CRISPR-CISH: an in situ chromogenic DNA repeat detection system for research and life science education

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月22日

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  • Bhanu Prakash Potlapalli,
  • Fabian Dassau,
  • Jörg Fuchs,
  • Deboprio Roy Sushmoy,
  • Andreas Houben

摘要

In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.