Biochemists rationalize the method of building human artificial chromosomes

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Biochemists rationalize the method of building human artificial chromosomes

For 20 years, researchers have been trying to perfect the construction of human artificial chromosomes, or HACs for short. In an article published today in Cell, Penn's researchers describe a new way to form an essential part of the artificial chromosome, called the centromere, by bypassing the biological requirements necessary to form a natural one. In simple terms, they biochemically delivered a protein called CENP-A directly to the HAC DNA to simplify the construction of a HAC in the laboratory.

"Our developments streamline the construction and characterization of HACs to help in efforts to manufacture whole synthetic human chromosomes," said Ben Black, PhD, professor of biochemistry and biophysics at the Perelman School of Medicine at the University of Pennsylvania, who has spent decades understanding this process.

HACs essentially function as new mini-chromosomes carrying sets of genes that are inherited along with the natural set of chromosomes in a cell. Bioengineers are considering that HACs can perform all kinds of tasks, including the delivery of large proteins for gene therapy or the transport of suicidal genes to fight cancer.

"Think of the HACs that we are now building as chromosomes the size of a model," said first author Glennis Logsdon, PhD, a PhD student in Black's laboratory at the time of the study and now a postdoctoral fellow at the University of Washington. "By building a centromere on a HAC in a simpler way, we are closer to obtaining normal size chromosomes."

The heredity of HACs from mother to daughter cells during division is essential, reflecting the importance of the centromere - the curved area of duplicated chromosomes responsible for maintaining the pairs of "brother" chromosomes created when cells divide. Without it, entire chromosomes can be lost during cell division.

In order for cell replication to occur, human centromers are not simply encoded by a DNA sequence, unlike research on synthetic baker's yeast chromosomes that has long been used. For example, mammals depend on the CENP-A protein to specify the location of the centromere on chromosomes for a specific cell division.

Previous attempts to form HAC centromeres in specimens have only rarely occurred when they have "encountered" A-CENP, and this unlikely event has only occurred at highly repetitive DNA sequences on the HAC genome. "However, highly repetitive DNA is the scourge of molecular biologists because it is the most difficult to work with the approaches we currently have, which are designed for non-repetitive DNA," said Dr. Black.

Black's team completely bypassed the repetitive DNA by delivering CENP-A directly to the HAC DNA. Their workaround solution is to "force" the CENP-A to combine with non-repetitive DNA sequences to form a new centromere for HAC.

"We used our centromere derivation method to obtain a fully functional AHC without the cloning nightmares that the centromere's repetitive DNA has presented to mammalian chromosome engineers over the past two decades," said Black. "Based on our success, we and others in the field of synthetic chromosomes will now have a real chance to achieve what has not been achieved so far in yeast cells."

One of the next steps in this field of synthetic biology will be to link the centromere of the black laboratory to gene sets that others have developed. This step-by-step construction project is the objective of the Human Genome Project - Writing, a collaboration to build this life-size synthetic chromosome. The contribution of the Penn team will accelerate the creation of useful clinical and research tools based on synthetic chromosomes.