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Understanding the three-dimensional organization of genomes within chromatin is essential for uncovering the mechanisms of gene regulation. Here, we present a protocol combining an ALFA-tagged dCas9 protein with an Arabidopsis thaliana centromere-specific gRNA and a fluorescence-labeled NbALFA nanobody to visualize centromeric sequences in fixed Arabidopsis thaliana nuclei. The use of a dCas9 protein carrying multiple ALFA-tags, together with a minibody and a fluorescence-labelled anti-rabbit secondary antibody, led to enhanced target-specific signals. This CRISPR-FISH-based approach, integrated with ALFA-tag technology, enables efficient labeling of DNA repeats in fixed A. thaliana nuclei.

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Application of ALFA-Tag Signal Amplification to Expand the CRISPR-Based DNA Imaging Toolkit

  • 实验方案

  • 发表日期：

    2026年

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  • Bhanu Prakash Potlapalli,
  • Andreas Houben

摘要

Understanding the three-dimensional organization of genomes within chromatin is essential for uncovering the mechanisms of gene regulation. Here, we present a protocol combining an ALFA-tagged dCas9 protein with an Arabidopsis thaliana centromere-specific gRNA and a fluorescence-labeled NbALFA nanobody to visualize centromeric sequences in fixed Arabidopsis thaliana nuclei. The use of a dCas9 protein carrying multiple ALFA-tags, together with a minibody and a fluorescence-labelled anti-rabbit secondary antibody, led to enhanced target-specific signals. This CRISPR-FISH-based approach, integrated with ALFA-tag technology, enables efficient labeling of DNA repeats in fixed A. thaliana nuclei.