Method for visually marking genome sites

Abstract

The invention discloses a method for visually marking genome sites. According to the method, a novel visual genome marking tool TTALE is established by fusing thioredoxin (TRX) at the c-terminal of the traditional transcriptional activation-like effect element (TALE) protein. Experiments prove that the TTALE can be used for accurately marking genome repetitive sequences such as telomere, centromere and ribosomal ribonucleic acid (RNA) coded sequences (ribosomal deoxyribonucleic acid (rDNA)) in different types of human cells such as a tumor cell line, embryonic stem cells, adult stem cells and terminally differentiated cells, and coded genetic loci (MUC4). A TTALE technology can make up for the lack of accurate, simple, convenient and long-acting live cell genome visualization marking techniques in the current scientific research and clinical markets, thereby promoting the development of basic research and clinical diagnosis and treatment of human senility and vital diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for visually marking genome sites. Background technique [0002] In the nucleus of human cells, about 3.2 billion base pairs form the huge human genome, which is further condensed to form 23 pairs of chromosomes of different sizes. Although the completion of the Human Genome Project has helped us obtain all the sequence information of the human genome, this is only for us to understand the structure of the human genome, and understand the three-dimensional structure of chromatin composed of the genome and important biological processes such as human development, aging and disease The first step in a relationship. In fact, many studies have proved that genome structure disorder is an important factor leading to aging and several serious diseases (Zhang, W., et al., Aging stem cells. A Werner syndrome stem cell model unveils heterochromatinalterations as a driver ...

AI Technical Summary

Problems solved by technology

Although a variety of genome visualization labeling technologies have been used in scientific research, the fluorescent labeling Lac or Tet system needs to integrate a large exogenous fragment of about 10kb into the target site of the genome, so there are problems such as low labeling efficiency and potential genome damage ( Robinett,C.C.,etal.,In vivo localization of DNA sequences andvisualization of large-scale chromatin organization using lac operator / repressor recognition.J Cell Biol,1996.135(6Pt 2):p.1685-700.2.Heun,P.,etal. ,Chromosome dynamics in the yeast interphase nucleus.Science,2001.294(5549):p.2181-6.); Fluorescence in situ hybridization (FISH) is currently the gold standard for studying the location of specific sequences in the genome, but this method requires chemical The operation is performed in fixed cells, so genome visualization in living cells cannot be achieved (Levsky, J.M. and R.H. Singer, Fluorescence in situ hybridization: past, present and future. J Cell Sci, 2003.116 (Pt 14): p.2833-8 .); Although the CRISPR / Cas9 technology that has emerged in recent years can achieve precise labeling of special genomic sites such as telomeres in living cells, the high background in the nucleus leads to low signal-to-noise ratios and complex systems. Accurate labeling of genomic loci in other human cells (such as pluripotent stem cells and terminally differentiated cells) other than cell lines (Levsky, J.M. and R.H. Singer, Fluorescence in situ hybridization: past, present and future. J Cell Sci , 2003.116(Pt 14): p.2833-8.); Another transcription activator-like effector element (TALE) used as gene editing technology can also be used for visual labeling of genomic loci, however existing Reports neither validated the correctness of TALE-mediated genomic labeling with the FISH gold standard, but also showed strong heterogeneity of labeling in different cell types (Ma, H., P. Reyes-Gutierrez, and T. Pederson, Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors.Proc NatlAcad Sci USA, 2013.110(52):p.21048-53.)

[0004] In summary, the existing genome visualization labeling technologies are flawed and cannot meet the needs of accurately labeling human cell genome elements and special sites

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