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

ABSTRACT: 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

SUBMITTER: Schubert MG 

PROVIDER: S-EPMC8106316 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature