Visualizing Nucleic Acid Loci with the CRISPR-Cas System: Current Approaches

Gerel A. A. Abushinova
Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation
Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation

Victoria V. Zherdeva orcid
Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation

Ekaterina M. Vassina orcid
Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation

Liliya G. Maloshenok orcid (Login required)
Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russian Federation
Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation


Paper #9176 received 9 Oct 2024; revised manuscript received 27 Nov 2024; accepted for publication 27 Nov 2024; published online 29 Dec 2024.

DOI: 10.18287/JBPE24.10.040203

Abstract

The visualization of genomic loci in living cells is crucial for detecting mutations and observing the spatial proximity of DNA regions in the 3D nuclear environment. One of the primary applications of the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR/Cas9) system is the non-invasive, real-time labeling of DNA loci in living cells, enabled by its unique characteristics. Visualization of genomic loci has been made possible by the use of an endonuclease-inactive form of the Cas9 protein (dCas9) and sgRNA in combination with fluorescent molecules. However, using CRISPR/Cas9 for targeting DNA regions has certain limitations, the most significant being suboptimal signal-to-noise ratio, the need for multiplexed labeling, and the large size of the Cas fluorescent reporter sytem, which impacts the complex’s functionality and complicates its delivery. Current variations of the method using CRISPR/dCas9 overcome these limitations in different ways. This review examines the evolution of genome locus visualization methods based on CRISPR/Cas9 from the initial use of the system to the present.

Keywords

CRISPR/Cas9; genome locus visualization; endonuclease-inactive Cas9 protein; nucleic acids

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