Design of CRISPR/Cas9 system components for visualization of specific genomic editing sites in tumor cell lines

Abstract

Most of the existing studies aimed at visualizing the loci of genome and determining the distance between them mainly rely on methods that provide information only on fixed cells and at the certain point in time. However, when using a modified CRISPR/Cas9 variant with an inactivated dCas9 protein that does not introduce breaks in DNA, but retains ability to bind to guide RNA and target DNA, it is possible to label DNA sequences in a living cell, and therefore in dynamics. To label chromatine loci, dCas9 protein are fused with fluorescent proteins and introduce in cell along with guide RNA. However, this method also have its own difficult such as background fluorescence. The signal-to-background ratio can be increased by using dCas9-FP pairs with one bright fluorescent protein and the best nuclear localization of the chimeric fluorescent molecule. Also, one of the ways to solve this problem is to create stable cell lines, for example, using lentiviral transduction using the Tet-ON inducible expression system. Different combinations of dCas9-FP orthologs show different localization of chimeric molecules in the cell. Localization of combinations of dCas9 orthologs chimeras from such organisms as Neisseria meningitidis, Streptococcus pyogenes and Streptococcus thermophillus and fluorescent proteins EGFP and mCherry were tested in the work, as well as ability of selected samples with the best nuclear localization to label repetitive telomeric sequences using guide RNA.
The work was carried out with the support of the RNF 22-14-00205

Speaker

Gerel Abushinova
The Federal State Institution “Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences”, Moscow; Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow
Russia

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