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High-throughput functional variant screens via in vivo production of single-stranded DNA
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High-throughput functional variant screens via in vivo production of single-stranded DNA
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High-throughput functional variant screens via in vivo production of single-stranded DNA
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Journal Article
High-throughput functional variant screens via in vivo production of single-stranded DNA
2021
Overview
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.
Publisher
National Academy of Sciences,Proceedings of the National Academy of Sciences
Subject
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ DNA
/ DNA, Single-Stranded - biosynthesis
/ DNA, Single-Stranded - genetics
/ Drug Resistance, Microbial - genetics
/ Genome, Bacterial - genetics
/ Genomes
/ Genomics
/ High-Throughput Nucleotide Sequencing
/ High-Throughput Screening Assays
/ retron
/ Saccharomyces cerevisiae - genetics
