Friday, April 25, 2025
4/25/2025
Self-Evolvr: self-refreshing molecular barcodes using CRISPR-self-guided mutagenesis - ABSTRACT DNA barcoding involves embedding unique, identifiable sequences of DNA into cells, which can then be sequenced at much higher depth than is possible across the genome as a whole, allowing tracing of evolutionary dynamics and lineages of those cells or other genetic elements over time. However, fixed barcode systems fail after the first clonal expansion or takeover, where the expansion of a population of genetically identical cells yields identical barcodes, meaning those individuals and their independent lineages are no longer distinguishable. This project develops Self-EvolvR, a new self-refreshing DNA barcode technology aimed at addressing the limitations of current evolving barcode systems for high-resolution lineage tracing across various biological systems. The research will focus on the development, optimization, and validation of Self-EvolvR to enable scalable, quantitative measurements of cellular lineages over time across a large variety of organisms and systems. Our approach uses an engineered Cas9 system to continually to introduce mutations into the barcode over long periods without self-inactivation. This method is designed to track both rapid and long-term cellular dynamics with a single construct, improving lineage resolution and reducing toxicity to the system. Aim 1 will construct the Self-EvolvR system to function in living cell lines, and validate the technology in vivo through lineage tracing in both bacterial and eukaryotic models, demonstrating its application across different biological systems. Aim 2 is to enhance the mutation rate of Self-EvolvR using directed evolution to achieve a resolution capable of distinguishing between cells separated by a few division events. The Self-EvolvR technology is designed to offer a more versatile and less toxic option in systems where barcodes can be introduced before the population dynamics of interest occur, facilitating new insights into cellular evolution and differentiation processes. The technology developed in this project will result in a new experimental tool for high-resolution lineage tracing applicable in a range of research fields including but not limited to microbiome studies, development, and immunology.
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