ENGINEERING CHROMOSOME MANIPULATION TECHNIQUES FOR ARTIFICIAL KARYOTYPE CONSTRUCTION IN MAMMALIAN CELLS

Abstract

Background: Techniques for chromosome manipulation have transformed synthetic genomics and mammalian genome engineering by allowing the assembly of artificial chromosomes and the design of designer karyotypes. Sophisticated chromosome engineering technologies provide powerful means to study genome organization, gene regulation and chromosomal stability in mammalian cells.

Objective: The purpose of this study was to review the current techniques of chromosome manipulation for artificial karyotype construction in mammalian systems and to assess their biomedical and synthetic biology applications.
Methods: We comparatively studied chromosome engineering strategies including CRISPR-Cas9 genome editing, microcell-mediated chromosome transfer (MMCT), synthetic chromosome assembly, and human artificial chromosome (HAC) technologies. Mammalian cell models were subjected to chromosome rearrangement, synthetic assembly, and genome stability evaluation by fluorescence in situ hybridization (FISH), sequencing, and karyotype analysis.

Findings:  Genome editing accuracy was around 80–92% using artificial chromosome engineering systems, with stable chromosome maintenance in >85% of the engineered cell populations. CRISPR-assisted chromosome manipulation greatly enhanced the efficiency of chromosomal rearrangement and the stability of the genome in the formation of an artificial karyotype.

Conclusion: Recent advances in chromosome engineering technologies show great promises for the applications in synthetic genome construction, disease modeling, regenerative medicine and gene therapy. Artificial karyotype engineering could further promote future precision synthetic biology and programmable mammalian genome design.