De novo assembly and delivery to mouse cells of a 101 kb functional human gene

被引：30

作者：

Mitchell, Leslie A. ^{[1
,2
,6
]}

McCulloch, Laura H. ^{[1
,2
]}

Pinglay, Sudarshan ^{[1
,2
]}

Berger, Henri ^{[1
,2
,3
]}

Bosco, Nazario ^{[1
,2
]}

Brosh, Ran ^{[1
,3
]}

Bulajic, Milica ^{[4
]}

Huang, Emily ^{[1
,3
]}

Hogan, Megan S. ^{[1
,3
]}

Martin, James A. ^{[1
,2
,7
]}

Mazzoni, Esteban O. ^{[4
]}

Davoli, Teresa ^{[1
,2
]}

Maurano, Matthew T. ^{[1
,3
]}

Boeke, Jef D. ^{[1
,2
,5
]}

机构：

[1] NYU Langone Hlth, Inst Syst Genet, New York, NY 10016 USA

[2] NYU Langone Hlth, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA

[3] NYU Langone Hlth, Dept Pathol, New York, NY 10016 USA

[4] NYU, Dept Biol, New York, NY 10003 USA

[5] NYU, Dept Biomed Engn, Tandon Sch Engn, Brooklyn, NY 11201 USA

[6] Opentrons Inc, New York, NY USA

[7] Weill Cornell Grad Sch Med Sci, New York, NY USA

来源：

GENETICS | 2021年 / 218卷 / 01期

关键词：

synthetic genomics; recombinase-mediated cassette exchange; genome writing; mammalian genome engineering; INDUCIBLE CASSETTE EXCHANGE; EMBRYONIC STEM; DNA FRAGMENTS; SCRAMBLE; YEAST; DISRUPTION; GENERATION; VIRUS;

D O I：

10.1093/genetics/iyab038

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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 workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

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