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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2017)

The directed differentiation of stem cells has a wide range of applications in fields such as regenerative medicine, in vitro cell biology, and disease modeling. Here, I present a CRISPR-based strategy to modulate target gene expression and direct cell fate. My system employs orthogonal nuclease-deactivated dCas9s derived from distinct bacterial species that when fused to transcriptional activator or repressor domains, enable the simultaneous activation and repression of specific target genes. By stably integrating these activating and repressing dCas9s into mouse embryonic stem cells (ESCs) to regulate the expression of specific transcription factors, I demonstrate an optimized strategy for the production of neurons. I show that this system increases the efficiency of neuronal induction relative to activation alone when genes that promote non-neuronal fates are simultaneously repressed. Additionally, using a different set of sgRNAs, I show that this system can be used to direct ESCs to a muscle cell fate. Altogether, my results establish a unique platform to program cell fate through the introduction of sgRNA cocktails to drive activation and repression of target genes.

    Cover page: Programmed cell fate changes in embryonic stem cells using CRISPR technology
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2015)
    The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.
      Cover page: Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2015)
      Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
        Cover page: Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2019)
        Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.
          Cover page: Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2022)
          A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.
            Cover page: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro
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