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Method to assemble genomic DNA fragments or genes on Human Artificial Chromosome with regulated kinetochore using a multi-integrase system Nicholas C.O. Lee, Jung-Hyun Kim, Nikolai Petrov, Hee-sheung Lee, Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, and Natalay Kouprina ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00209 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017
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ACS Synthetic Biology
Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System
Nicholas C.O. Lee1*, Jung-Hyun Kim1*, Nikolai S. Petrov1, Hee-Sheung Lee1, Hiroshi Masumoto2, William C. Earnshaw3, Vladimir Larionov1, Natalay Kouprina1**
1
Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA
2
Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-
7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan 3
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland
Keywords human artificial chromosome, HAC, iterative integration system, IIS, DNA assembly, synthetic biology
*These authors contributed equally. ** To whom correspondence should be addressed. Tel: +1 301 496 7941; Fax: +1 301 480 2772; Email
[email protected] 1
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ACS Synthetic Biology
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ABSTRACT
The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: i) Cloning of large genomic segments; ii) Insertion of multiple DNA blocks at a precise location and iii) The capability to eliminate the assembled region from cells. In this study, we designed the Iterative Integration System (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with Human Artificial Chromosome (HAC) possessing a regulated kinetochore (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes: The potential to assemble an unlimited number of genomic DNA segments; A DNA assembly process that leave only a small insertion (
