Journal: bioRxiv

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

doi: 10.1101/2025.06.27.658158

Figure Lengend Snippet: A. A synthetic gene library is first generated and sequenced to map 20-nt barcodes to their corresponding DNA molecules. Barcodes associated with perfect sequences are selected as protospacers for targeting. Spacer sequences are then in vitro transcribed into sgRNA libraries, which are complexed with dCas9 to form ribonucleoprotein complexes (RNPs). These RNPs are incubated with the synthetic library, allowing dCas9 to bind targeted barcodes. Biotinylated dCas9 is captured using streptavidin-coated magnetic beads, followed by bead pulldown and washes to remove unbound, non-target sequences. Enriched DNA is PCR-amplified and sequenced via nanopore to assess target enrichment. B. Rank-ordered read abundance of 18 selected target barcodes from a >300, 000-barcode single-gene rfp library. Each dot represents a barcode, ranked by read count. Dark magenta lines denote target barcodes, with magenta numbers indicating barcode IDs. Asterisks mark the three target barcodes used in BAR-CAT optimizations shown in . Scatter plot comparing the fraction of barcode reads in the rfp library before (original) and after enrichment. Each blue dot represents a non-target barcode; each magenta dot represents a target barcode. The red dashed unity line denotes equal representation before and after enrichment, serving as a reference for assessing enrichment magnitude. D . Enrichment conditions are shown chronologically along the x-axis. The colored bar above the plots summarizes key iterative protocol changes, including bead wash volume, DNA input format (linear vs. supercoiled), and dCas9 denaturation method. Violin plots display the distribution of log 2 enrichment scores for off-target (blue) and target (magenta) barcodes. Shaded areas represent the interquartile range (25th–75th percentile), with horizontal bars indicating median enrichment. Percent dropouts are indicated in bar plots for off-target (blue) and target (magenta) barcodes. All barcodes with reduced abundance (log 2 enrichment < 0), including barcodes that drop out post-enrichment, are reported as % depleted. The pink condition (6× 50 μL) corresponds to the original proof-of-concept protocol (v0.1) and is linked to panel C by a blue arrow.

Article Snippet: To determine the optimal number of PCR cycles, qPCR reactions were prepared using 1 ng of library S2 amplicons, 1.25 µL of 10 µM biotinylated forward primer (P5_FWD_Biotin_NV), 1.25 µL of 10 µM biotinylated reverse primer (P7_REV_Biotin_NV) ( Table S2 ), 0.25 µL of 100× Biotium Thiazole Green (Thermo Fisher Scientific), 12.5 µL of 2× Q5 High-Fidelity Master Mix (NEB), and nuclease-free water to a final volume of 25 µL.

Techniques: Generated, In Vitro, Incubation, Magnetic Beads, Amplification