Carrier for in-vivo positioning mammal cell genome based on CRISPRCas9 system and application thereof

Abstract

The invention discloses a carrier for in-vivo positioning mammal cell genome based on a CRISPRCas9 system and an application thereof. The carrier comprises mutant dCas9 protein of Cas9 protein and guide RNA of a specific gene bound with the mutant dCas9 protein, the mutant dCas9 protein has a SunTag system, the SunTag system contains a GCN4 antigenic epitope determinant, high-brightness green fluorescin mNeonGreen is connected with a GCN4 antibody, and the GCN4 antibody and the GCN4 antigenic epitope determinant are combined. The carrier for in-vivo positioning mammal cell genome based on the CRISPRCas9 system has strong fluorescence intensity, can observe the expression condition of the specific gene at each period under living cell condition, dynamic change trend of the specific gene during whole interphase and a division stage can be obtained, and the cells cannot be killed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a carrier for in vivo positioning of mammalian cell genomes based on a CRISPR Cas9 system and an application thereof. Background technique [0002] Clustered, regularly interspaced short palindromic repeats CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated systems) is currently the most efficient genome editing system. There are five types of CRISPR / Cas systems. The Type II CRISPR-Cas system has been widely and efficiently used in gene editing at the cellular and biological levels. It consists of Cas9 nucleoprotein and a guide RNA with crRNA and tracrRNA functions. The Cas9 protein contains two conserved regions that cut the target sequence, namely the HNH and Ruvc domains; and there is a crRNA:tracrRNA scaffold at the 3' end of the guide RNA, which can consolidate the interaction between the guide RNA and the Cas9 protein. The CRISP...

AI Technical Summary

Problems solved by technology

[0004] The fluorescent protein in the CRISPR imaging system is mostly sfGFP, which can achieve a good labeling effect in the case of DNA sequences with many repetitive sequences, but the fluorescence intensity is relatively weak. If you use an ordinary fluorescence microscope to capture the fluorescence of the marker gene Signal, especially when capturing sfGFP fluorescent protein-labeled repeat sequences, the difficulty factor is relatively large, and the fluorescent signal can only be enhanced by connecting several fluorescent proteins in series, but this method will reduce the efficiency of gene labeling and increase the noise , can not completely solve the problem of weak fluorescent signal

At present, there is no fluorescent protein with particularly strong fluorescence signal applied to the CRISPR imaging system. If a fluorescent protein with strong fluorescence intensity is found and applied to this system, it is believed that the changes of specific genes in each period can be observed more clearly and clearly. Probe the dynamic interactions of intrachromosomal and interchromosomal domains during cell cycle progression, during epigenetic regulation, or in response to cellular stimuli

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