Characterization of a CRISPR/Cas9-based molecular recorder for HIV-1
Latent human immunodeficiency virus type 1 (HIV-1) reservoirs in resting CD4+ T cells and myeloid-lineage cells such as macrophages and microglia remain the principal barrier to a cure, as antiretroviral therapy suppresses replication without eliminating integrated proviruses. These integrated proviruses are the main barrier to a cure. Current CRISPR-based therapeutic strategies as a cure for HIV-1 largely rely on endpoint measurements that cannot capture the temporal dynamics of viral activation and genome-editing activity over time. This thesis developed HIV-SCRIBE, a CRISPR-based molecular recorder in which a self-targeting guide RNA (stgRNA) locus is placed under a Tat-responsive minimal HIV-1 5'LTR promoter, coupling Cas9-mediated cleavage and error-prone repair to Tat-driven transcriptional activation to generate a durable molecular record of HIV-1 reactivation and, more broadly, of cell-type-specific CRISPR editing dynamics. The main objective of this thesis was to evaluate this molecular recorder system's ability to record Tat-dependent HIV-1 reactivation in HEK293T cells. Edits to the recorder, along with Cas9 protein and stgRNA production, were assessed at 48-hour, 72-hour, and one-week time points. Cas9 protein and stgRNA were detected at all time points examined; however, sequence analysis of recorder edits showed minimal editing across all time points, including one low-frequency variant, an insertion near the PAM site, that could itself limit further editing. RT-PCR further showed that although Tat was produced at each time point, it did not induce stgRNA expression. Future directions will need to determine whether the minimal promoter is functional in order to utilize the molecular recorder system across multiple cell types as originally designed.