Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Seeland, Germany.
Arid Land Research Center (ALRC), Tottori University, Hamasaka, Tottori, Japan.
Methods Mol Biol. 2023;2672:315-335. doi: 10.1007/978-1-0716-3226-0_20.
Fluorescence in situ hybridization (FISH) has been widely used to visualize target DNA sequences in fixed chromosome samples by denaturing the dsDNA to allow complementary probe hybridization, thus damaging the chromatin structure by harsh treatments. To overcome this limitation, a CRISPR/Cas9-based in situ labeling method was developed, termed CRISPR-FISH. This method is also known as RNA-guided endonuclease-in situ labeling (RGEN-ISL). Here we present different protocols for the application of CRISPR-FISH on acetic acid: ethanol or formaldehyde-fixed nuclei and chromosomes as well as tissue sections for labeling repetitive sequences in a range of plant species. In addition, methods on how immunostaining can be combined with CRISPR-FISH are provided.
荧光原位杂交（FISH）已被广泛用于通过使 dsDNA 变性以允许互补探针杂交来可视化固定染色体样品中的靶 DNA 序列，从而通过苛刻的处理破坏染色质结构。为了克服这一限制，开发了一种基于 CRISPR/Cas9 的原位标记方法，称为 CRISPR-FISH。该方法也称为 RNA 引导的内切酶原位标记（RGEN-ISL）。在这里，我们展示了在醋酸：乙醇或福尔马林固定的核和染色体以及组织切片上应用 CRISPR-FISH 的不同方案，用于标记多种植物物种中的重复序列。此外，还提供了如何将免疫染色与 CRISPR-FISH 结合使用的方法。
