title: A method for high-throughput production of sequence-verified DNA libraries and strain collections.
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.
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Justin D
authors.identifier: 0000-0003-3079-3534
authors.initials: JD
authors.lastname: Smith
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schlecht
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA.
authors.forename: Weihong
authors.initials: W
authors.lastname: Xu
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Sundari
authors.initials: S
authors.lastname: Suresh
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Joe
authors.initials: J
authors.lastname: Horecka
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Proctor
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Raeka S
authors.identifier: 0000-0002-3007-8963
authors.initials: RS
authors.lastname: Aiyar
authors.affiliation: Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA.|Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Richard A O
authors.initials: RA
authors.lastname: Bennett
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Chu
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
authors.forename: Yong Fuga
authors.initials: YF
authors.lastname: Li
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Roy
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.|European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Steinmetz
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Hyman
authors.affiliation: Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA.|Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Sasha F
authors.identifier: 0000-0002-0923-1636
authors.initials: SF
authors.lastname: Levy
authors.affiliation: Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA bstonge@stanford.edu.|Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Robert P
authors.identifier: 0000-0003-1626-3107
authors.initials: RP
authors.lastname: St Onge
mesh_terms: D064113:CRISPR-Cas Systems; D019295:Computational Biology / Q000379:methods; D004271:DNA, Fungal / Q000235:genetics; D015723:Gene Library; D017422:Sequence Analysis, DNA / Q000379:methods*; D015003:Yeasts / Q000235:genetics*
chemical_list: D004271:DNA, Fungal
keywords: CRISPR interference; DNA libraries; arrayed strain collection; oligonucleotide pools; synthetic biology