REVIEW: Visualizing the Nucleome Using the CRISPR–Cas9 System: From in vitro to in vivo

---

Liliya G. Maloshenok^1,2, Gerel A. Abushinova^1,2, Alexandra Yu. Ryazanova¹, Sergey A. Bruskin^1,2, and Victoria V. Zherdeva^1,a*

¹Bach Institute of Biochemistry, Federal Research Center for Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia

²Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received August 26, 2022; Revised September 30, 2022; Accepted October 3, 2022

---

One of the latest methods in modern molecular biology is labeling genomic loci in living cells using fluorescently labeled Cas protein. The NIH Foundation has made the mapping of the 4D nucleome (the three-dimensional nucleome on a timescale) a priority in the studies aimed to improve our understanding of chromatin organization. Fluorescent methods based on CRISPR–Cas are a significant step forward in visualization of genomic loci in living cells. This approach can be used for studying epigenetics, cell cycle, cellular response to external stimuli, rearrangements during malignant cell transformation, such as chromosomal translocations or damage, as well as for genome editing. In this review, we focused on the application of CRISPR–Cas fluorescence technologies as components of multimodal imaging methods for in vivo mapping of chromosomal loci, in particular, attribution of fluorescence signal to morphological and anatomical structures in a living organism. The review discusses the approaches to the highly sensitive, high-precision labeling of CRISPR–Cas components, delivery of genetically engineered constructs into cells and tissues, and promising methods for molecular imaging.

---

KEY WORDS: CRISPR–Cas9, fluorescent probes, 4D nucleome, chromosome loci, molecular imaging

DOI: 10.1134/S0006297923140080

---