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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genomics

Background:

• Array-synthesized oligonucleotide libraries offer low costs but suffer from high error rates and uneven representation.
• Limitations in accessing individual oligonucleotides hinder the full potential of these libraries in biological research.

Purpose of the Study:

• To develop a cost-effective method for creating high-purity and uniformly represented oligonucleotide libraries.
• To demonstrate the utility of this method for generating individually accessible strain collections.

Main Methods:

• Integration of complex oligonucleotide libraries into yeast.
• Site-specific recombination for indexing DNA.
• Next-generation sequencing for clone identification and characterization.

Main Results:

• Generated a DNA probe library of ~3,300 sequences with >96% purity and >95% uniformity.
• Created a collection of ~9,000 individually accessible CRISPR interference yeast strains.
• Achieved >99% coverage for genes essential for fermentative or respiratory growth.

Conclusions:

• Recombinase Directed Indexing (REDI) provides a rapid and cost-effective approach for generating and characterizing complex DNA libraries.
• The REDI method fundamentally improves the parsing, maintenance, propagation, and characterization of DNA libraries.
• This technique is adaptable for various applications in synthetic and quantitative biology.