سیستم گزارشگر غنی سازی سلولهای مثبت ویرایش ژنوم
Abstract
1- Introduction
2- Genome repair mechanisms
3- Intracellular free exogenous reporter system
4- Cell genome integrated endogenous reporter system
5- Outlooks
References
Abstract
Genome editing has become a vital tool in medical biology research. The critical mission of facilitating the progress of genome editing is to enrich different genes into positive edited cells quickly and effectively and carry out the targeted research. In recent years, researchers have established various reporter systems for selection and enrichment of editing-induced positive cells, which are based on genome repair mechanisms, such as non-homologous end joining, homology-directed repair, single strand annealing and inversion, and the principle of the expression of fluorescent protein or resistance tag after genome repair. The T7E1 assay or sequencing method can analyze the mutation of enriched cells with the results of lower background signals and higher mutation ratio. Therefore, these reporter systems can profit the characterization of genome editing effectiveness. Besides, positive cells can be cultured continuously, so this technology possesses a promising prospect in mutated cell line construction and the research of mutated cell functions. This article summarized the design principles and applications of these reporter systems and would provide a reference to construct a more perfecte evaluating system for genome editing.
Introduction
Genetic engineering started when monkey virus SV40 and E. coli bacteriophage λDNA spliced into a loop for the first time[1]. Subsequently, the discovery and research of genome repair mechanisms such as homologous recombination (HR), non-homologous end joining (NHEJ), and endonucleases provided a theoretical basis for modern genome editing technology[2,3]. In recent years, researchers have found some nucleases based on long sequence recognition, such as meganuclease[4‒7] or homing endonuclease, zinc finger nucleases (ZFNs)[8‒12], transcription activator-like effector nucleases (TALENs)[13‒17], structure-guided nucleases (SGN)[18] and clustered regularly interspaced short palindromic repeats associated protein Cas9 (CRISPR/Cas9)[19‒22]. These nucleases can accurately recognize the target sequence and induce double-strand break (DSB), and then through homologydirected repair (HDR)[23] or NHEJ[24] and other mechanisms to edit the target sequence. Genome editing technology has been applied in various fields, such as the establishment of disease models[26‒28], gene therapy[29], and gene screening[30] through different transfection techniques, such as liposomes, electroporation, viral packaging, and transmembrane peptides[25]. However, the off-target effects inherent in genome editing tools[31,32] may induce side effects such as non-target editing, erroneous phenotypes, or lethality in practical applications.