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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2014)

Cytoplasmic dynein is one of the principle motors in eukaryotic cells, responsible for most microtubule (MT) minus end-directed motility and force generation processes, including vesicular and organelle transport, and mitotic spindle MT organization. Despite being essential to the maintanence of cellular structure in higher eukaryotes, many aspects of dynein's stepping mechanism have remained enigmatic. In this Dissertation, I directly observe several fundamental aspects of dynein's mechanism, and build a strong framework for future studies. I outline in detail the methods for the fluorescent tracking of motor proteins, including advanced two-color and super-resolution techniques. To study cytoplasmic dynein, I developed a two-color fluorescent tracking assay to simultaneously measure the positions of both heads of the motor while it walks along the MT. The results of this experiment clearly show that dynein steps through a unique mechanism: the heads remain widely separated, and do not appear to coordinate with each other to a significant extent. However, the leading head is less likely to take a step at large inter-head separations, indicating some degree of residual coordination remains. I hypothesized that tension along the linker connecting the two heads is the source of this coordination. Using a high-speed two-color assay, I examined the motility of a homodimeric dynein with a flexible linker between the two heads that decreases inter-molecular tension. Despite this alteration, this homodimer has identical stepping and overall motility properties to wild-type dynein, but has reduced coordination, indicating that coordination is dispensable to motility. Coordination could be achieved by gating one of the heads. I investigated gating using a heterodimeric dynein with a wild-type head and an inactive, tightly-bound head. In this heterodimer, we find that the WT head is gated when widely separated from its inactive partner, but becomes un-gated when immediately behind it, indicating that the gating is caused by extension-dependent changes in linker tension as predicted. I next I turned from inter- to intra-molecular communication. Dynein has six distinct AAA subdomains on its motor head, of which only two AAA1 and AAA3, are essential to robust motility. By reducing MT affinity with added salt, I found that AAA3 is required for robust MT release, and that MT release in turn promotes hydrolysis at AAA1. To investigate coordination between AAA1 and AAA3, I analyzed motility in the presence of a slowly-hydrolyzable ATP analog, ATPγS. I found that ATPγS selectively inhibits the AAA3 site. In the presence of saturating analog, the motor can still take long runs of fast motility, indicating that the hydrolysis cycle of AAA3 is much shorter than AAA1. These results show that AAA1 and AAA3 do not coordinate. Instead, AAA3 acts as a "switch" that controls AAA1-directed release from the MT.

    Cover page: Studies of Inter-and Intra-Molecular Coordination in Cytoplasmic Dynein
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2014)
    Cytoplasmic dynein is a molecular motor responsible for minus-end-directed cargo transport along microtubules (MTs). Dynein motility has previously been studied on surface-immobilized MTs in vitro, which constrains the motors to move in two dimensions. In this study, we explored dynein motility in three dimensions using an MT bridge assay. We found that dynein moves in a helical trajectory around the MT, demonstrating that it generates torque during cargo transport. Unlike other cytoskeletal motors that produce torque in a specific direction, dynein generates torque in either direction, resulting in bidirectional helical motility. Dynein has a net preference to move along a right-handed helical path, suggesting that the heads tend to bind to the closest tubulin binding site in the forward direction when taking sideways steps. This bidirectional helical motility may allow dynein to avoid roadblocks in dense cytoplasmic environments during cargo transport.
      Cover page: Bidirectional helical motility of cytoplasmic dynein around microtubules
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5' fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering.
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2020)
        The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.
          Cover page: Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2017)
          Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.
            Cover page: Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2019)
            Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2019;37:284-294.
              Cover page: Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2016)
              Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.
                Cover page: Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells
                • Article
                • Peer Reviewed
                Berkeley Research Impact Initiative (BRII) (2019)
                Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as ?-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential ?-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated ?-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in ?-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of ?-globin. These data suggest that the 1.7 kb region is not an autonomous ?-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2017)
                  CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2022)
                    Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.
                      Cover page: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
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