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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2021)

It is projected that by the year 2050, global agriculture will need to increase the output of staple crops by 60% to feed the world’s rapidly growing population amidst threats posed by climate change and crop disease. One strategy to meet this challenge is to develop new genetic diversity in the germplasm to generate robust and resilient lines with beneficial traits. Conventional breeding has played an essential and major role in crop trait improvement. However, those methods can be difficult, laborious, and time-consuming. The development of effective technology for precision and time-saving plant breeding is required. A breakthrough tool to this end has been CRISPR-Cas9. Noteworthy for its simple sequence-specific programmability, the gene editing system has unleashed remarkable potential for plant biotechnology and functional genomics. Here, we explore several aspects and parameters to advance Cas9-mediated gene editing technology in the essential monocot food crops: rice and wheat. Early-stage plant gene-editing projects benefit greatly from a rapid transient pipeline using protoplasts to evaluate the efficacy of gene editing reagents. We describe here a novel and sustainable method for protoplast isolation from rice tissue and demonstrate their use in ribonucleoprotein (RNP) based Cas9 gene editing assays. Next, we focus on applications of Cas9 technology in wheat to engineer disease resistance. Wheat is a critical target organism because the complexity of its large allohexaploid genome has rendered genetic manipulation by classic methods extremely difficult. Using DNA plasmids encoding CRISPR-Cas9 gene editing reagents, we optimize conditions and demonstrate that all three homoeologous copies of genes in allohexaploid wheat can be simultaneously edited within a single generation. We recognize, however, that DNA plasmid gene editing approaches have a number of limitations including random integration into the plant genome and unpredictability of expression. To address this, we finally present significant improvements to DNA-free Cas9-RNP based gene editing in wheat. We show that increased temperature treatment greatly enhances Cas9-mediated editing efficiency and regenerate transgene-free edited wheat plants at a high rate. Lastly, we utilize this DNA-free gene editing method to generate de novo partial resistance to the agronomically important pathogen Parastagonospora nodorum.

    Cover page: Advancing enabling technology and genome editing in monocot crops for disease resistance and sustainability
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2020)

    Background

    An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.

    Results

    Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.

    Conclusion

    The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
      Cover page: Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2014)
      The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
        Cover page: Selection of chromosomal DNA libraries using a multiplex CRISPR system
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2018)
        Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.
          Cover page: Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host
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