Modern gene synthesis platforms enable investigations of protein function and genome biology at an unprecedented scale. Yet, the proportion of error-free constructs in diverse gene libraries decreases with length due to the propagation of oligo synthesis errors. To rescue these error-free constructs, we developed Barcode-Assisted Retrieval CRISPR-Activated Targeting (BAR-CAT), an in vitro method that uses multiplexed dCas9-single-guide RNA (sgRNA) complexes to extract barcodes corresponding to error-free constructs. After a 15-min incubation and wash regimen, three low-bundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to a 384-gene DropSynth gene library, BAR-CAT enriched 12 targets up to 122-fold and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.
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