Mendelian inheritance of artificial chromosomes

Synthetic mammalian artificial chromosomes (MACs) represent a new frontier in genome technology,
with the potential to transform chromosome and synthetic biology and stimulate the development of numerous
radical advances in medicine. Human Genome Project-Write aims to generate an entire set of synthetic human
chromosomes. Short of this ambitious goal, MACs have enormous potential for breakthroughs in biotechnology
and medicine, such as creating humanized animal models for drug development or for harvesting patient-
personalized organs for transplantation. Furthermore, building MACs from minimal components will advance
our fundamental understanding of what comprises a mammalian chromosome.
As vehicles for genetic inheritance, fully functional chromosomes are faithfully transmitted through
mitosis and the specialized meiotic divisions underlying eukaryotic sexual reproduction and Mendelian
inheritance. Our goal is to construct the first MACs that achieve faithful inheritance through the germline, using
mouse as a model system. One obstacle is the centromere, the locus on each chromosome that directs
transmission through both mitosis and meiosis. Because mammalian centromeres are not encoded in the DNA
sequence, it is unclear how to build synthetic chromosomes containing this crucial element. There are
additional challenges to create MACs that pair and recombine as homologous chromosomes in meiosis. To
solve these problems, we will hijack the existing cellular machinery for assembling centromere chromatin and
incorporate additional genetic elements to ensure meiotic pairing and recombination.
This effort requires innovation at multiple levels: designing MAC vectors encoding key functional
elements, assembling large synthetic DNA constructs, and ultimately creating animals to test MACs in vivo.
The proposed work builds on recent advances from the co-investigators’ teams in all of these areas, and we
have key tools and expertise in place to build the necessary DNA templates, introduce them into embryos,
analyze the outcomes, and develop alternative strategies as necessary. The most meaningful preliminary data
would be to show a synthetic artificial chromosome that is successfully transmitted through mitosis and meiosis
in vivo, but achieving this step is a major goal of our proposal and will require substantial investment of time
and effort. Thus, we are requesting support for this project without the preliminary data that would demonstrate
high likelihood of success, justifying consideration of our proposal as part of the T-R01 mechanism.
We use mouse as a relatively rapid and tractable mammalian model system with outstanding
opportunities for testing and debugging MACs, and our advances should readily transfer to other species for
applications in biotechnology and medicine. Success in this project will represent a quantum leap in the
development of synthetic artificial chromosome that are fully functional in vivo, providing unprecedented
genome engineering capabilities in animal models and enabling diverse synthetic biology applications.

Grant Number: 5R01HG012445-05
NIH Institute/Center: NIH
Principal Investigator: Ben Black