Post-Acquisition Enrichment of Protospacer Containing Plasmids Using CcdB Counterselection in Record-Seq

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.