High-throughput functional variant screens via in vivo production of single-stranded DNA

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasse...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2021-05, Vol.118 (18), p.1-10
Main Authors: Schubert, Max G., Goodman, Daniel B., Wannier, Timothy M., Kaur, Divjot, Farzadfard, Fahim, Lu, Timothy K., Shipman, Seth L., Church, George M.
Format: Article
Language:English
Subjects:
Alleles
Antibiotic resistance
Antibiotics
BASIC BIOLOGICAL SCIENCES
Biological Sciences
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
DNA, Single-Stranded - biosynthesis
DNA, Single-Stranded - genetics
Drug Resistance, Microbial - genetics
Escherichia coli - genetics
functional genomics
Gene Library
Genetic diversity
Genetic Engineering
Genetic variance
Genome, Bacterial - genetics
Genomes
Genomics
Growth rate
High-Throughput Nucleotide Sequencing
High-Throughput Screening Assays
Nucleotide sequence
Phenotyping
retron
Saccharomyces cerevisiae - genetics
Science & Technology - Other Topics
Single-stranded DNA
Synthetic Biology
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Summary:Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2018181118