Publications by authors named "Owen W Ryan"

7 Publications

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Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.

mBio 2018 09 25;9(5). Epub 2018 Sep 25.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

Throughout history, the yeast 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, 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 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 can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish as a synthetic biology platform comparable to , with naturally more robust traits that hold potential for the industrial production of renewable chemicals. The yeast 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 in industrial applications. Here, we describe genetic tools for genome editing and breeding strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using as a versatile synthetic biology platform organism for industrial applications.
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http://dx.doi.org/10.1128/mBio.01410-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156195PMC
September 2018

CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

Cold Spring Harb Protoc 2016 06 1;2016(6). Epub 2016 Jun 1.

Energy Biosciences Institute, University of California, Berkeley, California 94720; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; Department of Chemistry, University of California, Berkeley, California 94720.

This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.
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http://dx.doi.org/10.1101/pdb.prot086827DOI Listing
June 2016

Multiplex engineering of industrial yeast genomes using CRISPRm.

Methods Enzymol 2014 ;546:473-89

Energy Biosciences Institute, University of California, Berkeley, California, USA; Department of Molecular and Cell Biology, University of California, Berkeley, California, USA; Department of Chemistry, University of California, Berkeley, California, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. Electronic address:

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.
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http://dx.doi.org/10.1016/B978-0-12-801185-0.00023-4DOI Listing
July 2015

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

Elife 2014 Aug 19;3. Epub 2014 Aug 19.

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.

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.
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http://dx.doi.org/10.7554/eLife.03703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161972PMC
August 2014

A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1.

Mol Cell 2003 Dec;12(6):1565-76

Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.

Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects. Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1. The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails. Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway. We show that recruitment of Htz1 to chromatin requires the SWR-C. Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.
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http://dx.doi.org/10.1016/s1097-2765(03)00497-0DOI Listing
December 2003

A panoramic view of yeast noncoding RNA processing.

Cell 2003 Jun;113(7):919-33

Banting and Best Department of Medical Research, University of Toronto, 112 College Street, M5G 1L6, Toronto, Ontario, Canada.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.
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http://dx.doi.org/10.1016/s0092-8674(03)00466-5DOI Listing
June 2003

The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation.

Mol Cell 2003 Mar;11(3):721-9

Banting and Best Department of Medical Research, Department of Molecular and Medical Genetics, University of Toronto, Toronto, M5G 1L6, Ontario, Canada.

Methylation of histone proteins is one of their many modifications that affect chromatin structure and regulate gene expression. Methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively, is required for silencing of expression of genes located near chromosome telomeres in yeast. We report that the Paf1 protein complex, which is associated with the elongating RNA polymerase II, is required for methylation of lysines 4 and 79 of histone H3 and for silencing of expression of a telomere-associated gene. We show that the Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II and that the subunits of these complexes interact physically and genetically. Collectively, our results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation.
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http://dx.doi.org/10.1016/s1097-2765(03)00091-1DOI Listing
March 2003