UC Irvine

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell’s genome, thereby allowing the hidden forces that guide tissue development to be chronicled. After the developmental process of interest is complete, the cells’ experiences can be read out via high-throughput DNA sequencing. Two types of transient events are captured with DNA recorders—the existence of progenitor cells that give rise to daughter cells (which is recorded as lineage relationships among cells by generating lineage-specific DNA barcodes), and stimuli that trigger a molecular response in the cells (which is recorded in an analog fashion by linking the accumulation of mutations to the presence of inducers of interest). This dissertation focuses on the development of two distinct, but complementary DNA recorders. First, Cell History Recording by Ordered iNsertion (CHYRON), which uses a homing guide RNA (hgRNA), CRISPR-Cas9, and a template-independent polymerase (TdT) to generate ordered insertion mutations, was developed. A large portion of this technology was already established prior to my joining the project, so this dissertation focuses only on the developments that overlapped with my efforts. Specifically, alternative nuclease components (instead of Cas9) were explored for CHYRON recording, and dose- and duration-dependent hypoxia recording was accomplished. Also, the nucleotide insertion preferences of TdT were engineered in a novel, massively parallel screen, to achieve greater lineage barcode diversity and enable recording of two different stimuli with differently-biased TdTs. The other DNA recorder covered in this work is prime editing CHYRON (peCHYRON), which uses prime editor to generate iterative insertion mutations. Prime editor requires its edits to be precisely pre-defined in prime editing guide RNAs (pegRNAs), so pegRNA sequences that accomplish continuous insertion accumulation were carefully engineered via a combination of rational design and high-throughput screening. Once continuous editing was embodied by peCHYRON, we used the system for in vitro cell lineage tracing and molecular stimulus recording. Efforts to expand peCHYRON’s ability to record a massive collection of stimuli (i.e., any stimulus that can induce transcription with RNAP II) are discussed.