Thesis Format
Monograph
Degree
Master of Science
Program
Biochemistry
Supervisor
Edgell, David
Abstract
CRISPR is the adaptive immune system in archaea and prokaryotes which has garnered attention for its genome editing applications. Beyond genome editing, CRISPR-Cas has been explored for recording transcriptional activity, addressing gaps in current technologies and offering insights into complex microbial communities. Record-Seq is one such technology that encodes transcriptional activity as DNA protospacers stored in a CRISPR array within a recorder plasmid. However, low protospacer acquisition efficiency limits the broader application of Record-Seq. To address this, we developed a post-recording enrichment strategy leveraging the potent toxin CcdB to selectively enrich recorder plasmids containing protospacers. This approach involved placing ccdB downstream of the CRISPR array, such that protospacer insertion would disrupt the reading frame, rendering CcdB non-functional and allowing cell survival. Despite its theoretical feasibility, our analysis revealed that this strategy failed to enrich for protospacers. These findings underscore the challenges in post-recording enrichment and highlight opportunities for advancing transcriptional recorder technologies.
Summary for Lay Audience
CRISPR is the adaptive immune system in archaea and prokaryotes and is widely known for its genome editing applications. These genome editing applications utilize CRISPR-Cas proteins that are directly involved in destroying pathogens. Recently, CRISPR-Cas proteins involved in recording pathogenic events have been repurposed to create transcriptional recorders. Record-Seq leverages these recording CRISPR-Cas proteins to convert RNA into DNA protospacers that are inserted into a CRISPR array. These protospacers are sequentially stored, creating a cumulative record of transcripts expressed within a cell. However, the low protospacer acquisition efficiency of recording CRISPR-Cas proteins necessitates intensive screening, limiting the applicability of Record-Seq.
The focus of my thesis was to reduce the need for extensive protospacer screening by selectively eliminating cells that did not acquire protospacers. To address this, we implemented a post-recording enrichment strategy using the CcdB counterselection method, which has been previously used for similar enrichment purposes. Despite our efforts, we were unable to successfully enrich for protospacers. Our work explores the challenges of post-recording enrichment using CcdB and proposes alternative strategies to enhance emerging transcriptional recording technologies.
Recommended Citation
Sharath, Keerthana, "Post-Acquisition Enrichment of Protospacer Containing Plasmids Using CcdB Counterselection in Record-Seq" (2025). Electronic Thesis and Dissertation Repository. 10847.
https://ir.lib.uwo.ca/etd/10847
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.