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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2016)

More than 40 million people world-wide suffer from tauopathies–neurodegenerative diseases in which the aggregation of microtubule-associated protein tau into intracellular inclusions is a core pathology. Recent studies have shown that the ubiquitin-proteasome system (UPS) and the autophagy pathway are responsible for the normal turnover of tau in neurons. Autophagy is a conserved cellular mechanism that is essential for neuronal homeostasis and impaired as a result of aging, which is the primary risk factor for most tauopathies. Thus, we hypothesized that dysfunctions in the autophagy pathway impair turnover of tau, and thus facilitate pathogenic aggregation. To test this hypothesis, we first treated human induced pluripotent stem cell (hiPSC)-derived neurons with two compounds that have been shown to promote autophagy, and found that these compounds decreased levels of phosphorylated tau protein, suggesting that autophagy may mediate homeostatic clearance of phospho-tau in human neurons. We also found that inhibition of the UPS induces autophagy and promotes the clearance of both phospho-tau and total tau non-selectively.

In order to identify whether autophagy truly regulates the clearance of phosphorylated tau, we set out to establish a CRISPR/Cas9-based system that would allow us to disrupt autophagy in an efficient and inducible fashion. We have characterized two inducible systems, gathering a foundational understanding of how the system can be applied to genetic screens in hiPSC-derived cell types. Thus, this study both expands on the current knowledge regarding degradation of tau in human neurons and lays the groundwork for further genetic studies using an inducible-Cas9 system.

    Cover page: Elucidating the role of autophagy in the clearance of phosphorylated tau protein in human neurons
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2018)
    Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.
      Cover page: Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2018)
      CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
        Cover page: Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2023)
        Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.
          Cover page: Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2021)
          Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.
            Cover page: Barcoded viral tracing of single-cell interactions in central nervous system inflammation
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