September 22, 2019  |  

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Authors: Mitchell, Leslie A. and McCulloch, Laura H. and Pinglay, Sudarshan and Berger, Henri and Bulajic, Milica and Martin, James A. and Hogan, Megan S. and Mazzoni, Esteban and Maurano, Matthew T. and Boeke, Jef D.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger, mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assemble the 101 kb human HPRT1 gene in yeast, deliver it to mouse cells, and show expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells.

Journal: BioRxiv
DOI: 10.1101/423426
Year: 2018

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