Constructing human artificial chromosomes (HACs) in budding yeast overcomes the long-standing problem of uncontrolled multimerization – the rampant joining of similar molecules – and results in HACs that are large, stable, and structurally well-defined, researchers report. The findings may help advance chromosome engineering for precise genome editing in mammals and many other organisms.

Artificial chromosomes can carry large numbers of engineered genes. Their use in bacteria and yeast as vehicles for writing and rewriting genomes has hinted at their potential to provide an alternative approach to editing genetic material in human cell lines.

Although the first HACs were developed nearly 25 years ago, the multimerization and uncontrolled rearrangement of the input DNA during HAC formation has greatly limited their usefulness for precise genome engineering in humans.

Here, the researchers present a new approach to constructing HACs, which avoids unintended multimerization. The HAC produced by the authors was initially assembled as a circular artificial chromosome in budding yeast and transferred into the human cell line by passing the chromosome directly from the yeast into cultured cells using cell fusion.

According to the authors, the newly formed HACs rarely multimerized after being transferred to human cells and instead maintained as single-copy molecules. Compared to prior HACs, the ones constructed by the authors are much larger, ~750 kilobase pairs of DNA, which is large enough to house the multi-domain chromatin required for inheritance through cell divisions.

“Future applications of HACs will likely focus on introducing long genes or multigene clusters into cell lines or individuals,” writes a researcher in a related Perspective. “It may soon be possible to include artificial chromosomes as a part of an expanding toolkit to address global challenges related to healthcare, livestock, and the production of food and fiber.”

https://www.science.org/doi/10.1126/science.adj3566

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