Park Eui-Jin, Kim Hajin
Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.
Exp Mol Med. 2025 Jul;57(7):1392-1399. doi: 10.1038/s12276-025-01498-x. Epub 2025 Jul 31.
CRISPR-Cas-based genome imaging opened a new era of genome visualization in living cells. While genomic loci with repetitive sequences, such as centromeres and telomeres, can be reliably imaged, applying the technique to nonrepetitive genomic loci has remained challenging. Recent advancements in the design of CRISPR RNAs and Cas proteins, the development of novel fluorophores and the combination of CRISPR-Cas with other molecular machinery amplified target-specific signals and suppressed background signals, revolutionizing this unique genome imaging technique and enabling the tracking of genomic loci with a small number of CRISPR-Cas complexes, down to a single complex. Here we review the latest advancements in CRISPR-Cas-based genome imaging techniques and their application to imaging nonrepetitive genomic loci. The challenges that these techniques are currently facing are the cellular toxicity and genomic instability induced by the expression of CRISPR-Cas and its interference with DNA metabolism, which impacts DNA replication and genome maintenance. Recently reported adverse effects of CRISPR-Cas-based genome labeling are discussed here, along with perspectives on how to overcome the problem.
基于CRISPR-Cas的基因组成像开启了活细胞中基因组可视化的新时代。虽然具有重复序列的基因组位点，如着丝粒和端粒，可以可靠地成像，但将该技术应用于非重复基因组位点仍然具有挑战性。CRISPR RNA和Cas蛋白设计的最新进展、新型荧光团的开发以及CRISPR-Cas与其他分子机制的结合放大了靶标特异性信号并抑制了背景信号，彻底改变了这种独特的基因组成像技术，并能够用少量的CRISPR-Cas复合物追踪基因组位点，甚至低至单个复合物。在这里，我们回顾了基于CRISPR-Cas的基因组成像技术的最新进展及其在非重复基因组位点成像中的应用。这些技术目前面临的挑战是CRISPR-Cas表达诱导的细胞毒性和基因组不稳定性，以及它对DNA代谢的干扰，这会影响DNA复制和基因组维护。这里讨论了最近报道的基于CRISPR-Cas的基因组标记的不良反应，以及关于如何克服该问题的观点。
