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Mol Syst Biol. 2017 Feb 13;13(2):913. doi: 10.15252/msb.20167233.

A method for high-throughput production of sequence-verified DNA libraries and strain collections.

Author information

1
Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
2
Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
3
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
4
Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA.
5
Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA.
6
Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY, USA.
7
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
8
Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA bstonge@stanford.edu.

Abstract

The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.

KEYWORDS:

CRISPR interference; DNA libraries; arrayed strain collection; oligonucleotide pools; synthetic biology

PMID:
28193641
PMCID:
PMC5327727
DOI:
10.15252/msb.20167233
[Indexed for MEDLINE]
Free PMC Article

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