BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment

Abstract: Modern gene-synthesis platforms let us probe protein function and genome biology at unprecedented scale. Yet in large, diverse gene libraries the proportion of error-free constructs decreases with length due to the propagation of oligo synthesis errors. To rescue these rare, error-free molecules we developed BAR-CAT (Barcode-Assisted Retrieval CRISPR-Activated Targeting), an in-vitro enrichment method that couples unique PAM-adjacent 20-nt barcodes to each library member and uses multiplexed dCas9-sgRNA complexes to fish out the barcodes corresponding to perfect assemblies. After a single 15-min reaction and optimized wash regime (BAR-CAT v1.0), three low-abundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to 384x and 1,536x member DropSynth gene libraries, BAR-CAT retrieved up to 122-fold enrichment for 12 targets 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 versatile platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.This dataset contains processed enrichment data for all spacers including:sequence - spacer sequencestatus - if the sequence was an enrichment target or notlog2enrich - log2 fold enrichment scorereads.ini - initial reads before enrichmentreads.postenrich - reads after enrichmentThe raw Illumina MiSeq reads of the target gene libraries and nanopore sequence reads for enriched libraries are available on the NCBI Sequence Read Archive under BioProject accession PRJNA1273454 (https://www.ncbi.nlm.nih.gov/bioproject/1273454).