Repetitive DNA, defined as sequence motifs that recur throughout the genome, comprises about 54% of the human genome. Large amounts of repetitive DNA were long labeled “junk” or “selfish” DNA, yet growing evidence shows that repeats in both coding and noncoding regions play essential roles in evolution, gene regulation, and genetic variation, and more than 60 diseases, most of them neurological, are caused by repeat expansion. Long-read sequencing and bioinformatics now allow these regions to be characterized in depth, but engineering repetitive DNA remains mainly limited by repeat instability.
Recently, the group led by Prof. Yi Wu at Tianjin University, published a review article, “Technologies for engineering repetitive DNA,” in
Quantitative Biology. The review provides a comprehensive overview of current strategies for constructing repetitive DNA:
in vitro approaches, including isothermal amplification, mispairing-guided polymerase extension, restriction endonuclease-mediated ligation, in vitro homologous recombination, and redundancy-minimizing design, can generate repeat tracts of kilobases to tens of kilobases;
in vivo methodologies in
Escherichia coli,
Saccharomyces cerevisiae, and mammalian cells assemble longer and more complex arrays, with amplification editing achieving precise duplications from 20 bp to 100 Mb. The authors further discuss how these methods enable multiplex CRISPR-Cas systems, artificial chromosome engineering, models of repeat expansion diseases, and the synthesis of protein-based and nucleic acid biomaterials, and they call for a shift from circumventing repeat instability toward actively regulating it.
Figure 1 illustrates the overall framework of repetitive DNA engineering. Engineering strategies, both
in vitro and
in vivo, are organized around the construction of repetitive DNA and feed into four application areas: models of repetitive DNA, biomaterial synthesis, multiplexed CRISPR-Cas systems, and artificial chromosomes. Looking ahead, the authors argue that host strain engineering, epigenetic control of repeat accessibility, and AI-guided design could turn repeat stability into a tunable parameter. They foresee that “a shift from ‘difficult to synthesize’ toward ‘precisely controllable’ could improve the manufacturability and predictability of complex repeat constructs, providing a firmer methodological footing for higher-order artificial genomes and synthetic life systems.”
DOI
10.1002/qub2.70040