Technologies for engineering repetitive DNA

Abstract Repetitive DNA, a fundamental architectural element of genomes, is widespread across organisms and comprises about 54% of the human genome. With advances in long‐read sequencing and bioinformatics approaches, highly repetitive sequences can now be characterized in depth. Nevertheless, engineering repetitive DNA is mainly limited by repeat instability. Despite these challenges, advances have been reported in the construction of short tandem repeats and the large‐scale duplication of genes. This review provides a comprehensive overview of current strategies for constructing repetitive DNA, covering both in vitro approaches, including restriction endonuclease ligation, in vitro homologous recombination, and redundancy‐minimizing sequences, as well as in vivo methodologies in Escherichia coli , Saccharomyces cerevisiae , and mammalian cells. We further discuss applications of these methods for developing genetic manipulation tools, constructing models of repetitive DNA, and synthesizing biomaterials. Future progress in engineering repetitive DNA will further advance our capabilities in designing and manipulating complex biological systems and deepen our understanding of them.