Innovations in Programmable Nucleic Acid Libraries and CRISPR Enrichment for Molecular Biology Applications

Synthetic gene libraries are pivotal in advancing protein engineering, functional genomics, and synthetic biology, yet their quality is hindered by errors in oligonucleotide synthesis. These errors pose significant challenges for large-scale gene synthesis of long genes due to excessive imperfect assemblies. This dissertation presents innovative methods that leverage CRISPR-Cas9 technologies for targeted retrieval of perfect gene assemblies, aiming to increase their length and quality.A major contribution of this work is the development of Barcode Assisted Retrieval-CRISPR Activated Targeting (BAR-CAT), a method that uses deactivated Cas9 (dCas9) to selectively enrich perfect synthetic genes from complex libraries. By tagging genes with unique DNA barcodes and targeting these barcodes with in vitro transcribed sgRNAs, we successfully enriched three targeted barcodes by up to 1,094-fold. However, BAR-CAT scalability was limited beyond 12 targeted barcodes due to challenges with excessive library diversity and competition among sgRNAs for dCas9 binding, which will require further method optimization. Parallel to the development of BAR-CAT, I led the development of a scalable sgRNA synthesis workflow that reduces costs by over 70% by harnessing large pools of microarray-derived oligos. These oligos are assembled into dsDNA templates and in vitro transcribed to generate sgRNA libraries. Despite optimizations, RNA-seq analysis revealed biases in spacer representation driven by guanine-rich sequences near the T7 promoter. We mitigated these biases by padding sgRNA spacers with a guanine tetramer and evaluating alternative approaches, such as compartmentalization in emulsions. These strategies improved sgRNA library uniformity, with broad implications for CRISPR-Cas9 screens and sgRNA design. Taken together, these advances in targeted DNA enrichment and sgRNA library production are advances that will contribute to improving the scalability, affordability, and fidelity of synthetic gene libraries. BAR-CAT, in particular, offers a promising approach for multiplexed retrieval of perfect genes that could benefit applications in synthetic biology, ancient DNA analysis, diagnostics, and targeted sequencing. Future refinements to both BAR-CAT and sgRNA synthesis methods will further extend their utility, enabling high-throughput exploration of protein function and genome biology. This dissertation includes unpublished co-authored material.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Department of Biology

Grantor dc:publisher

University of Oregon

Year dc:date.issued

2025

Author dc:creator

• Villegas, Natanya

Advisor dc:contributor.advisor

• Plesa, Calin

• CRISPR-Cas9
• DNA synthesis
• DropSynth
• Gene Synthesis
• in vitro transcription
• single guide RNAs

Statement dc:rights

• All Rights Reserved.

Language dc:language.iso

en_US

Handle dc:identifier.uri

https://hdl.handle.net/1794/31514

OAI record oai:identifier

https://scholarsbank.uoregon.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ascholarsbank.uoregon.edu%3A1794%2F31514