Biology subjects

Villegas, N. K.

Publications and source records attributed to Villegas, N. K..

3 recordsLinked to original sources

bioRxiv2026

DropSynth-Gold: Golden Gate Assembly in Emulsions Extends Multiplexed Gene Libraries to Greater Lengths

The ability to synthesize longer genes at scale remains a central challenge in multiplexed gene synthesis. DropSynth is a pooled gene synthesis platform that enables highly multiplexed, compartmentalized assembly from microarray-derived oligonucleotides, but current implementations rely on polymerase cycling assembly (PCA), which constrains fragment number, construct length, and assembly fidelity. Here we present DropSynth-Gold, an evolution of the DropSynth platform that replaces PCA with Golden Gate assembly (GGA) performed within emulsion droplets. This modification preserves the core workflow, including bead-linked oligonucleotide capture and pooled processing, while altering only the assembly chemistry and computational oligo design strategy. Emulsion-based Golden Gate assembly enables directional, multi-fragment ligation within isolated droplets, followed by recovery and amplification of full-length constructs. As a proof of concept, we constructed six 384-member libraries spanning increasing construct lengths and fragment counts, including designs from 5x300-mer fragments to 12x350-mer architectures ([~]3 kb). DropSynth-Gold reliably assembled full-length constructs across all libraries. A direct comparison of a shared 5x300-mer library demonstrated comparable recovery and fidelity to PCA-based DropSynth, indicating that Golden Gate assembly can replace PCA without compromising assembly performance. These gains were achieved without increasing cost, workflow complexity, or turnaround time, expanding the accessible design space for multiplexed gene synthesis.

synthetic biology↗

bioRxiv2025

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

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.

synthetic biology↗

bioRxiv2025

Optimizing in-vitro Transcribed CRISPR-Cas9 single-guide RNA Libraries for Improved Uniformity and Affordability

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer, immediately downstream of the T7 promoter, as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.

molecular biology↗