Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides.

Abstract:

:Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands of genomic sites simultaneously. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.

journal_name

ACS Synth Biol

journal_title

ACS synthetic biology

authors

Bonde MT,Kosuri S,Genee HJ,Sarup-Lytzen K,Church GM,Sommer MO,Wang HH

doi

10.1021/sb5001565

subject

Has Abstract

pub_date

2015-01-16 00:00:00

pages

17-22

issue

1

issn

2161-5063

journal_volume

4

pub_type

信件
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