Precise gene modification mediated by TALEN and single-stranded oligodeoxynucleotides in human cells

Area of Science:

• * Stem cell biology
• * Gene editing technologies
• * Molecular genetics

Background:

• * Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are crucial for disease modeling, drug screening, and cell replacement therapy.
• * Efficient site-specific gene editing is essential for studying genotype-phenotype relationships in these cells.
• * Transcription activator-like effector nucleases (TALENs) offer a promising tool for gene editing due to their ease of assembly and high efficiency.

Purpose of the Study:

• * To optimize TALEN design for enhanced genomic cutting efficiency in human cells.
• * To demonstrate efficient gene editing using optimized TALENs combined with single-strand oligodeoxynucleotides (ssODNs).
• * To establish genetically modified cell lines for research into cell differentiation and tumorigenesis.

Main Methods:

• * Optimization of TALEN design to maximize DNA cutting efficiency.
• * Application of optimized TALENs with ssODNs for gene editing in human cells.
• * Creation of human tumor and H9 ESC lines with homozygous deletions of miR-21 and miR-9-2 genes.

Main Results:

• * Optimized TALENs achieved high-efficiency gene editing in human cells.
• * Successful gene mutations and deletions up to 7.8 kb were accomplished.
• * Homozygous deletion cell lines for miR-21 and miR-9-2 were generated, serving as a research platform.
• * Endogenous homologous chromosomes were observed to function as donor templates for gene editing.

Conclusions:

• * Optimized TALENs and ssODNs provide a versatile and efficient method for gene editing in human cells.
• * The established cell lines offer a valuable resource for studying gene function in differentiation and cancer.
• * This approach supports the development of genetically modified cells for research and potential therapeutic applications.