Potlapalli Bhanu Prakash, Dassau Fabian, Fuchs Jörg, Sushmoy Deboprio Roy, Houben Andreas
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstraße 3, 06466, Seeland, Germany.
Hochschule Mittweida, Department of Biotechnology, 09648, Mittweida, Germany.
Chromosome Res. 2025 Apr 22;33(1):7. doi: 10.1007/s10577-025-09767-1.
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.
原位杂交是一种用于可视化细胞核和染色体内特定DNA序列的技术。已经开发了多种DNA原位荧光标记方法，这些方法通常在探针杂交之前进行整体DNA变性，并且通常需要荧光显微镜来进行可视化。在此，我们报告了一种CRISPR/dCas9介导的显色原位DNA检测（CRISPR-CISH）方法的开发，该方法将显色信号检测与CRISPR成像相结合。这种非荧光方法使用3'生物素标记的反式激活CRISPR RNA（tracrRNA）和靶标特异性CRISPR RNA（crRNA）形成成熟的向导RNA（gRNA），从而激活dCas9以结合靶标序列。随后应用链霉亲和素碱性磷酸酶或辣根过氧化物酶会产生显色的、靶标特异性信号，这些信号可以使用传统的明场显微镜进行分析。此外，还确定了染色质复染剂，以帮助解释CRISPR-CISH产生的靶标信号。这一进展使原位DNA检测技术在资源有限的环境中更容易被研究人员、诊断应用和教育机构所使用。
