Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Jensen, Emil D.; Ferreira, Raphael; Jakočiūnas, Tadas; Arsovska, Dushica; Zhang, Jie; Ding, Ling; Smith, Justin D.; David, Florian; Nielsen, Jens; Jensen, Michael K.; Keasling, Jay D.

doi:10.1186/s12934-017-0664-2

Title: Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Abstract

Background Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site. Results In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker’s yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutivemore »« less

Authors:

Jensen, Emil D. ^[1]; Ferreira, Raphael ^[2]; Jakočiūnas, Tadas ^[1]; Arsovska, Dushica ^[1]; Zhang, Jie ^[1]; Ding, Ling ^[1]; Smith, Justin D. ^[3]; David, Florian ^[2]; Nielsen, Jens ^[4];

^[1]; Keasling, Jay D. ^[5]

The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark
Department of Biology and Biological Engineering, Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
Stanford University School of Medicine, Stanford, CA, (United States). Dept. of Genetics; Stanford Genome Technology Center, Palo Alto, CA, (United States)
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark; Dept. of Biology and Biological Engineering, Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark; Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United Staets). Dept. of Chemical and Biomolecular Engineering & Dept. of Engineering

Publication Date:: Wed Mar 15 00:00:00 EDT 2017

Research Org.:: Lawrence Berkeley National Lab (LBNL), Berkeley, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC); Novo Nordisk Foundation

OSTI Identifier:: 1626889

Grant/Contract Number:: AC02-05CH11231

Resource Type:: Accepted Manuscript

Journal Name:: Microbial Cell Factories

Additional Journal Information:: Journal Volume: 16; Journal Issue: 1; Journal ID: ISSN 1475-2859

Publisher:: BioMed Central

Country of Publication:: United States

Language:: English

Subject:: Biotechnology & Applied Microbiology

Citation Formats


                    Jensen, Emil D., Ferreira, Raphael, Jakočiūnas, Tadas, Arsovska, Dushica, Zhang, Jie, Ding, Ling, Smith, Justin D., David, Florian, Nielsen, Jens, Jensen, Michael K., and Keasling, Jay D. Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies.  United States: N. p., 2017. 
Web.  doi:10.1186/s12934-017-0664-2.

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                    Jensen, Emil D., Ferreira, Raphael, Jakočiūnas, Tadas, Arsovska, Dushica, Zhang, Jie, Ding, Ling, Smith, Justin D., David, Florian, Nielsen, Jens, Jensen, Michael K., & Keasling, Jay D. Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies.  United States.  https://doi.org/10.1186/s12934-017-0664-2

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                    Jensen, Emil D., Ferreira, Raphael, Jakočiūnas, Tadas, Arsovska, Dushica, Zhang, Jie, Ding, Ling, Smith, Justin D., David, Florian, Nielsen, Jens, Jensen, Michael K., and Keasling, Jay D. Wed .  
"Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies".  United States.  https://doi.org/10.1186/s12934-017-0664-2.  https://www.osti.gov/servlets/purl/1626889.

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@article{osti_1626889,

  title        = {Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies},

  author       = {Jensen, Emil D. and Ferreira, Raphael and Jakočiūnas, Tadas and Arsovska, Dushica and Zhang, Jie and Ding, Ling and Smith, Justin D. and David, Florian and Nielsen, Jens and Jensen, Michael K. and Keasling, Jay D.},

  abstractNote = {Background Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site. Results In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker’s yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG. Conclusion Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.},

  doi          = {10.1186/s12934-017-0664-2},

  journal      = {Microbial Cell Factories},

  number       = 1,

  volume       = 16,

  place        = {United States},

  year         = {Wed Mar 15 00:00:00 EDT 2017},

  month        = {Wed Mar 15 00:00:00 EDT 2017}

}

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Figures / Tables:

Fig. 1: Comparing two dCas9 systems for transcriptional regulation. a The anhydro-tetracyclin (aTc) inducible system with gRNA expression controlled by TetO reprograms transcriptional expression with dCas9 directly fused to either VPR or Mxi1 anchoring to promoters of genes of interest (GOI). b The constitutive dCas9 and scRNA expression system regulatesmore »

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Works referenced in this record:

Regulation of glycogen metabolism in yeast and bacteria
journal, November 2010

Wilson, Wayne A.; Roach, Peter J.; Montero, Manuel
FEMS Microbiology Reviews, Vol. 34, Issue 6
DOI: 10.1111/j.1574-6976.2010.00220.x

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development
journal, January 2014

Pajoro, Alice; Madrigal, Pedro; Muiño, Jose M.
Genome Biology, Vol. 15, Issue 3
DOI: 10.1186/gb-2014-15-3-r41

Large‐scale ¹³ C‐flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli
journal, January 2011

Haverkorn van Rijsewijk, Bart R. B.; Nanchen, Annik; Nallet, Sophie
Molecular Systems Biology, Vol. 7, Issue 1
DOI: 10.1038/msb.2011.9

A Comprehensive Genomic Binding Map of Gene and Chromatin Regulatory Proteins in Saccharomyces
journal, February 2011

Venters, Bryan J.; Wachi, Shinichiro; Mavrich, Travis N.
Molecular Cell, Vol. 41, Issue 4
DOI: 10.1016/j.molcel.2011.01.015

Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering
journal, March 2015

Alonso-Gutierrez, Jorge; Kim, Eun-Mi; Batth, Tanveer S.
Metabolic Engineering, Vol. 28
DOI: 10.1016/j.ymben.2014.11.011

Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay
journal, September 2013

Lee, Michael E.; Aswani, Anil; Han, Audrey S.
Nucleic Acids Research, Vol. 41, Issue 22
DOI: 10.1093/nar/gkt809

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode
journal, March 2012

Scalcinati, Gionata; Knuf, Christoph; Partow, Siavash
Metabolic Engineering, Vol. 14, Issue 2
DOI: 10.1016/j.ymben.2012.01.007

From flavors and pharmaceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae
journal, November 2013

Tippmann, Stefan; Chen, Yun; Siewers, Verena
Biotechnology Journal, Vol. 8, Issue 12
DOI: 10.1002/biot.201300028

Harnessing yeast subcellular compartments for the production of plant terpenoids
journal, September 2011

Farhi, Moran; Marhevka, Elena; Masci, Tania
Metabolic Engineering, Vol. 13, Issue 5
DOI: 10.1016/j.ymben.2011.05.001

Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae
journal, January 2012

Scalcinati, Gionata; Partow, Siavash; Siewers, Verena
Microbial Cell Factories, Vol. 11, Issue 1
DOI: 10.1186/1475-2859-11-117

Lipidomics: coming to grips with lipid diversity
journal, July 2010

Shevchenko, Andrej; Simons, Kai
Nature Reviews Molecular Cell Biology, Vol. 11, Issue 8
DOI: 10.1038/nrm2934

Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals
journal, November 2015

Jullesson, David; David, Florian; Pfleger, Brian
Biotechnology Advances, Vol. 33, Issue 7
DOI: 10.1016/j.biotechadv.2015.02.011

Biochemistry, cell biology and molecular biology of lipids ofSaccharomyces cerevisiae
journal, December 1998

Daum, Gunther; Lees, Norman D.; Bard, Martin
Yeast, Vol. 14, Issue 16
DOI: 10.1002/(SICI)1097-0061(199812)14:16<1471::AID-YEA353>3.0.CO;2-Y

Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae
journal, October 2016

Fang, Zhijia; Chen, Zhongxiang; Wang, Song
Applied and Environmental Microbiology, Vol. 83, Issue 1
DOI: 10.1128/AEM.02319-16

Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica
journal, May 2015

Qiao, Kangjian; Imam Abidi, Syed Hussain; Liu, Hongjuan
Metabolic Engineering, Vol. 29, p. 56-65
DOI: 10.1016/j.ymben.2015.02.005

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals
journal, January 2014

Runguphan, Weerawat; Keasling, Jay D.
Metabolic Engineering, Vol. 21
DOI: 10.1016/j.ymben.2013.07.003

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
journal, July 2013

Gilbert, Luke A.; Larson, Matthew H.; Morsut, Leonardo
Cell, Vol. 154, Issue 2, p. 442-451
DOI: 10.1016/j.cell.2013.06.044

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
journal, February 2013

Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A.
Cell, Vol. 152, Issue 5, p. 1173-1183
DOI: 10.1016/j.cell.2013.02.022

CRISPR RNA–guided activation of endogenous human genes
journal, July 2013

Maeder, Morgan L.; Linder, Samantha J.; Cascio, Vincent M.
Nature Methods, Vol. 10, Issue 10
DOI: 10.1038/nmeth.2598

SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae
journal, February 2017

Vanegas, Katherina García; Lehka, Beata Joanna; Mortensen, Uffe Hasbro
Microbial Cell Factories, Vol. 16, Issue 1
DOI: 10.1186/s12934-017-0632-x

Systematic testing of enzyme perturbation sensitivities via graded dCas9 modulation in Saccharomyces cerevisiae
journal, March 2017

Deaner, Matthew; Alper, Hal S.
Metabolic Engineering, Vol. 40
DOI: 10.1016/j.ymben.2017.01.012

Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design
journal, March 2016

Smith, Justin D.; Suresh, Sundari; Schlecht, Ulrich
Genome Biology, Vol. 17, Issue 1
DOI: 10.1186/s13059-016-0900-9

Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds
journal, January 2015

Zalatan, Jesse G.; Lee, Michael E.; Almeida, Ricardo
Cell, Vol. 160, Issue 1-2
DOI: 10.1016/j.cell.2014.11.052

Highly efficient Cas9-mediated transcriptional programming
journal, March 2015

Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani
Nature Methods, Vol. 12, Issue 4
DOI: 10.1038/nmeth.3312

CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing
journal, May 2014

Montague, Tessa G.; Cruz, José M.; Gagnon, James A.
Nucleic Acids Research, Vol. 42, Issue W1
DOI: 10.1093/nar/gku410

High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous
journal, May 2007

Verwaal, R.; Wang, J.; Meijnen, J. -P.
Applied and Environmental Microbiology, Vol. 73, Issue 13
DOI: 10.1128/AEM.02759-06

CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae
journal, March 2015

Jakočiu̅nas, Tadas; Rajkumar, Arun S.; Zhang, Jie
ACS Synthetic Biology, Vol. 4, Issue 11
DOI: 10.1021/acssynbio.5b00007

High-level semi-synthetic production of the potent antimalarial artemisinin
journal, April 2013

Paddon, C. J.; Westfall, P. J.; Pitera, D. J.
Nature, Vol. 496, Issue 7446
DOI: 10.1038/nature12051

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
journal, December 2014

Konermann, Silvana; Brigham, Mark D.; Trevino, Alexandro E.
Nature, Vol. 517, Issue 7536
DOI: 10.1038/nature14136

CRISPR-Cas9 Based Engineering of Actinomycetal Genomes
journal, April 2015

Tong, Yaojun; Charusanti, Pep; Zhang, Lixin
ACS Synthetic Biology, Vol. 4, Issue 9
DOI: 10.1021/acssynbio.5b00038

The DNA-encoded nucleosome organization of a eukaryotic genome
journal, December 2008

Kaplan, Noam; Moore, Irene K.; Fondufe-Mittendorf, Yvonne
Nature, Vol. 458, Issue 7236
DOI: 10.1038/nature07667

Nucleosomes impede Cas9 access to DNA in vivo and in vitro
journal, March 2016

Horlbeck, Max A.; Witkowsky, Lea B.; Guglielmi, Benjamin
eLife, Vol. 5
DOI: 10.7554/eLife.12677

Quantitative Analysis of Proteome and Lipidome Dynamics Reveals Functional Regulation of Global Lipid Metabolism
journal, March 2015

Casanovas, Albert; Sprenger, Richard R.; Tarasov, Kirill
Chemistry & Biology, Vol. 22, Issue 3
DOI: 10.1016/j.chembiol.2015.02.007

Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acc1
journal, May 2014

Shi, Shuobo; Chen, Yun; Siewers, Verena
mBio, Vol. 5, Issue 3
DOI: 10.1128/mBio.01130-14

The Yap family and its role in stress response
journal, May 2010

Rodrigues-Pousada, Claudina; Menezes, Regina A.; Pimentel, Catarina
Yeast, Vol. 27, Issue 5
DOI: 10.1002/yea.1752

Transcriptional Networks Controlling the Cell Cycle
journal, January 2013

Bonke, Martin; Turunen, Mikko; Sokolova, Maria
G3: Genes|Genomes|Genetics, Vol. 3, Issue 1
DOI: 10.1534/g3.112.004283

Metabolic engineering of Saccharomyces cerevisiae for linalool production
journal, November 2015

Amiri, Pegah; Shahpiri, Azar; Asadollahi, Mohammad Ali
Biotechnology Letters, Vol. 38, Issue 3
DOI: 10.1007/s10529-015-2000-4

CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production
journal, November 2016

Kim, Seong Keun; Han, Gui Hwan; Seong, Wonjae
Metabolic Engineering, Vol. 38
DOI: 10.1016/j.ymben.2016.08.006

Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR–Cas9-Mediated Genome Editing in Yarrowia lipolytica
journal, January 2016

Schwartz, Cory M.; Hussain, Murtaza Shabbir; Blenner, Mark
ACS Synthetic Biology, Vol. 5, Issue 4
DOI: 10.1021/acssynbio.5b00162

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch
journal, May 2016

Oakes, Benjamin L.; Nadler, Dana C.; Flamholz, Avi
Nature Biotechnology, Vol. 34, Issue 6
DOI: 10.1038/nbt.3528

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
journal, October 2015

Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.
Cell, Vol. 163, Issue 3, p. 759-771
DOI: 10.1016/j.cell.2015.09.038

Multidimensional chemical control of CRISPR–Cas9
journal, October 2016

Maji, Basudeb; Moore, Christopher L.; Zetsche, Bernd
Nature Chemical Biology, Vol. 13, Issue 1
DOI: 10.1038/nchembio.2224

EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9
journal, June 2016

Jessop-Fabre, Mathew M.; Jakočiūnas, Tadas; Stovicek, Vratislav
Biotechnology Journal, Vol. 11, Issue 8
DOI: 10.1002/biot.201600147

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae
journal, November 2010

Partow, Siavash; Siewers, Verena; Bjørn, Sara
Yeast, Vol. 27, Issue 11
DOI: 10.1002/yea.1806

Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae
journal, March 2015

Jakočiūnas, Tadas; Bonde, Ida; Herrgård, Markus
Metabolic Engineering, Vol. 28
DOI: 10.1016/j.ymben.2015.01.008

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
Nature Methods, Vol. 6, Issue 5, p. 343-345
DOI: 10.1038/nmeth.1318

A large-scale full-length cDNA analysis to explore the budding yeast transcriptome
journal, November 2006

Miura, F.; Kawaguchi, N.; Sese, J.
Proceedings of the National Academy of Sciences, Vol. 103, Issue 47
DOI: 10.1073/pnas.0605645103

Rapid Quantification of Yeast Lipid using Microwave-Assisted Total Lipid Extraction and HPLC-CAD
journal, May 2013

Khoomrung, Sakda; Chumnanpuen, Pramote; Jansa-Ard, Suwanee
Analytical Chemistry, Vol. 85, Issue 10
DOI: 10.1021/ac3032405

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.
text, January 2014

Pajoro, Alice; Madrigal, Pedro; Muiño, Jose M.
Apollo - University of Cambridge Repository
DOI: 10.17863/cam.54103

Transcriptional networks controlling the cell cycle.
text, January 2013

Bonke, Martin; Turunen, Mikko; Sokolova, Maria
Apollo - University of Cambridge Repository
DOI: 10.17863/cam.59547

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression
journal, February 2021

Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A.
Cell, Vol. 184, Issue 3
DOI: 10.1016/j.cell.2021.01.019

Large-scale 13C-flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli
text, January 2011

Haverkorn Van Rijsewijk, Bart R. B.; Nanchen, Annik; Nallet, Sophie
ETH Zurich
DOI: 10.3929/ethz-b-000162898

The DNA-encoded nucleosome organization of a eukaryotic genome
text, January 2009

Yair, Field,; D., Lieb, Jason; J., Gossett, Andrea
The University of North Carolina at Chapel Hill University Libraries
DOI: 10.17615/cste-0t39

From flavors and pharmaceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae
journal, November 2013

Tippmann, Stefan; Chen, Yun; Siewers, Verena
Biotechnology Journal, Vol. 8, Issue 12
DOI: 10.1002/biot.201300028

The Yap family and its role in stress response
journal, May 2010

Rodrigues-Pousada, Claudina; Menezes, Regina A.; Pimentel, Catarina
Yeast, Vol. 27, Issue 5
DOI: 10.1002/yea.1752

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae
journal, November 2010

Partow, Siavash; Siewers, Verena; Bjørn, Sara
Yeast, Vol. 27, Issue 11
DOI: 10.1002/yea.1806

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
journal, October 2015

Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.
Cell, Vol. 163, Issue 3, p. 759-771
DOI: 10.1016/j.cell.2015.09.038

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression
journal, February 2021

Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A.
Cell, Vol. 184, Issue 3
DOI: 10.1016/j.cell.2021.01.019

Quantitative Analysis of Proteome and Lipidome Dynamics Reveals Functional Regulation of Global Lipid Metabolism
journal, March 2015

Casanovas, Albert; Sprenger, Richard R.; Tarasov, Kirill
Chemistry & Biology, Vol. 22, Issue 3
DOI: 10.1016/j.chembiol.2015.02.007

A Comprehensive Genomic Binding Map of Gene and Chromatin Regulatory Proteins in Saccharomyces
journal, February 2011

Venters, Bryan J.; Wachi, Shinichiro; Mavrich, Travis N.
Molecular Cell, Vol. 41, Issue 4
DOI: 10.1016/j.molcel.2011.01.015

Harnessing yeast subcellular compartments for the production of plant terpenoids
journal, September 2011

Farhi, Moran; Marhevka, Elena; Masci, Tania
Metabolic Engineering, Vol. 13, Issue 5
DOI: 10.1016/j.ymben.2011.05.001

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode
journal, March 2012

Scalcinati, Gionata; Knuf, Christoph; Partow, Siavash
Metabolic Engineering, Vol. 14, Issue 2
DOI: 10.1016/j.ymben.2012.01.007

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals
journal, January 2014

Runguphan, Weerawat; Keasling, Jay D.
Metabolic Engineering, Vol. 21
DOI: 10.1016/j.ymben.2013.07.003

Systematic testing of enzyme perturbation sensitivities via graded dCas9 modulation in Saccharomyces cerevisiae
journal, March 2017

Deaner, Matthew; Alper, Hal S.
Metabolic Engineering, Vol. 40
DOI: 10.1016/j.ymben.2017.01.012

Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae
journal, January 2001

Kennedy, Matthew A.; Bard, Martin
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Vol. 1517, Issue 2
DOI: 10.1016/s0167-4781(00)00246-3

Rapid Quantification of Yeast Lipid using Microwave-Assisted Total Lipid Extraction and HPLC-CAD
journal, May 2013

Khoomrung, Sakda; Chumnanpuen, Pramote; Jansa-Ard, Suwanee
Analytical Chemistry, Vol. 85, Issue 10
DOI: 10.1021/ac3032405

CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae
journal, March 2015

Jakočiu̅nas, Tadas; Rajkumar, Arun S.; Zhang, Jie
ACS Synthetic Biology, Vol. 4, Issue 11
DOI: 10.1021/acssynbio.5b00007

The DNA-encoded nucleosome organization of a eukaryotic genome
journal, December 2008

Kaplan, Noam; Moore, Irene K.; Fondufe-Mittendorf, Yvonne
Nature, Vol. 458, Issue 7236
DOI: 10.1038/nature07667

CRISPR RNA–guided activation of endogenous human genes
journal, July 2013

Maeder, Morgan L.; Linder, Samantha J.; Cascio, Vincent M.
Nature Methods, Vol. 10, Issue 10
DOI: 10.1038/nmeth.2598

Machine learning discovery of missing links that mediate alternative branches to plant alkaloids
journal, March 2022

Vavricka, Christopher J.; Takahashi, Shunsuke; Watanabe, Naoki
Nature Communications, Vol. 13, Issue 1
DOI: 10.1038/s41467-022-28883-8

Endogenous promoter-driven sgRNA for monitoring the expression of low-abundance transcripts and lncRNAs
journal, January 2021

Gao, Ni; Hu, Jing; He, Bingbing
Nature Cell Biology, Vol. 23, Issue 1
DOI: 10.1038/s41556-020-00610-9

A large-scale full-length cDNA analysis to explore the budding yeast transcriptome
journal, November 2006

Miura, F.; Kawaguchi, N.; Sese, J.
Proceedings of the National Academy of Sciences, Vol. 103, Issue 47
DOI: 10.1073/pnas.0605645103

CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing
journal, May 2014

Montague, Tessa G.; Cruz, José M.; Gagnon, James A.
Nucleic Acids Research, Vol. 42, Issue W1
DOI: 10.1093/nar/gku410

Regulation of glycogen metabolism in yeast and bacteria
journal, November 2010

Wilson, Wayne A.; Roach, Peter J.; Montero, Manuel
FEMS Microbiology Reviews, Vol. 34, Issue 6
DOI: 10.1111/j.1574-6976.2010.00220.x

Synthesis of Triacylglycerols by the Acyl-Coenzyme A:Diacyl-Glycerol Acyltransferase Dga1p in Lipid Particles of the Yeast Saccharomyces cerevisiae
journal, January 2002

Sorger, D.; Daum, G.
Journal of Bacteriology, Vol. 184, Issue 2
DOI: 10.1128/jb.184.2.519-524.2002

Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae
journal, January 2012

Scalcinati, Gionata; Partow, Siavash; Siewers, Verena
Microbial Cell Factories, Vol. 11, Issue 1
DOI: 10.1186/1475-2859-11-117

SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae
journal, February 2017

Vanegas, Katherina García; Lehka, Beata Joanna; Mortensen, Uffe Hasbro
Microbial Cell Factories, Vol. 16, Issue 1
DOI: 10.1186/s12934-017-0632-x

Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design
journal, March 2016

Smith, Justin D.; Suresh, Sundari; Schlecht, Ulrich
Genome Biology, Vol. 17, Issue 1
DOI: 10.1186/s13059-016-0900-9

Transcriptional Networks Controlling the Cell Cycle
journal, January 2013

Bonke, Martin; Turunen, Mikko; Sokolova, Maria
G3: Genes|Genomes|Genetics, Vol. 3, Issue 1
DOI: 10.1534/g3.112.004283

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.
text, January 2014

Pajoro, Alice; Madrigal, Pedro; Muiño, Jose M.
Apollo - University of Cambridge Repository
DOI: 10.17863/cam.54103

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function
journal, April 2016

Isaac, R. Stefan; Jiang, Fuguo; Doudna, Jennifer A.
eLife, Vol. 5
DOI: 10.7554/elife.13450

Works referencing / citing this record:

Advancing Metabolic Engineering of Saccharomyces cerevisiae Using the CRISPR/Cas System
journal, April 2018

Lian, Jiazhang; HamediRad, Mohammad; Zhao, Huimin
Biotechnology Journal, Vol. 13, Issue 9
DOI: 10.1002/biot.201700601

CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces
journal, July 2018

Zhao, Yawei; Li, Lei; Zheng, Guosong
Biotechnology Journal, Vol. 13, Issue 9
DOI: 10.1002/biot.201800121

Whole‐Genome Regulation for Yeast Metabolic Engineering
journal, November 2019

Jiang, Shuangying; Si, Tong; Dai, Junbiao
Small Methods, Vol. 4, Issue 2
DOI: 10.1002/smtd.201900640

CRISPR/Cas9‐RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae
journal, May 2019

Kildegaard, Kanchana Rueksomtawin; Tramontin, Larissa Ribeiro Ramos; Chekina, Ksenia
Yeast, Vol. 36, Issue 5
DOI: 10.1002/yea.3390

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing
journal, September 2019

Payen, Celia; Thompson, Dawn
Yeast, Vol. 36, Issue 12
DOI: 10.1002/yea.3439

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis
journal, November 2019

Zhan, Yangyang; Xu, Yong; Zheng, Pengling
Applied Microbiology and Biotechnology, Vol. 104, Issue 1
DOI: 10.1007/s00253-019-10230-5

Marker-free genetic manipulations in yeast using CRISPR/CAS9 system
journal, April 2018

Soreanu, Inga; Hendler, Adi; Dahan, Danielle
Current Genetics, Vol. 64, Issue 5
DOI: 10.1007/s00294-018-0831-y

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape
journal, January 2018

Ferreira, Raphael; David, Florian; Nielsen, Jens
Journal of Industrial Microbiology & Biotechnology, Vol. 45, Issue 7
DOI: 10.1007/s10295-017-2000-6

Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system
journal, June 2019

Xu, Xiuwen; Cao, Xiaojuan; Gao, Jian
Transgenic Research, Vol. 28, Issue 3-4
DOI: 10.1007/s11248-019-00125-6

Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
journal, November 2017

Lian, Jiazhang; HamediRad, Mohammad; Hu, Sumeng
Nature Communications, Vol. 8, Issue 1
DOI: 10.1038/s41467-017-01695-x

Designing with living systems in the synthetic yeast project
journal, July 2018

Szymanski, Erika; Calvert, Jane
Nature Communications, Vol. 9, Issue 1
DOI: 10.1038/s41467-018-05332-z

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion
journal, January 2018

Ferreira, Raphael; Teixeira, Paulo Gonçalves; Siewers, Verena
Proceedings of the National Academy of Sciences, Vol. 115, Issue 6
DOI: 10.1073/pnas.1715282115

Multiplex genome editing of microorganisms using CRISPR-Cas
journal, April 2019

Adiego-Pérez, Belén; Randazzo, Paola; Daran, Jean Marc
FEMS Microbiology Letters, Vol. 366, Issue 8
DOI: 10.1093/femsle/fnz086

Enhanced scale and scope of genome engineering and regulation using CRISPR/Cas in Saccharomyces cerevisiae
journal, October 2019

Deaner, Matthew; Alper, Hal S.
FEMS Yeast Research, Vol. 19, Issue 7
DOI: 10.1093/femsyr/foz076

A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast
journal, November 2018

Hofmann, Anja; Falk, Johannes; Prangemeier, Tim
Nucleic Acids Research, Vol. 47, Issue 1
DOI: 10.1093/nar/gky1191

Gene repression via multiplex gRNA strategy in Y. lipolytica
journal, April 2018

Zhang, Jin-lai; Peng, Yang-Zi; Liu, Duo
Microbial Cell Factories, Vol. 17, Issue 1
DOI: 10.1186/s12934-018-0909-8

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC
journal, October 2018

Eng, Thomas; Demling, Philipp; Herbert, Robin A.
Microbial Cell Factories, Vol. 17, Issue 1
DOI: 10.1186/s12934-018-1006-8

Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha
journal, July 2018

Kurylenko, Olena O.; Ruchala, Justyna; Vasylyshyn, Roksolana V.
Biotechnology for Biofuels, Vol. 11, Issue 1
DOI: 10.1186/s13068-018-1203-z

Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae
journal, January 2018

Roggenkamp, Emily; Giersch, Rachael M.; Schrock, Madison N.
G3: Genes|Genomes|Genetics, Vol. 8, Issue 3
DOI: 10.1534/g3.117.300557

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains
journal, September 2017

Roggenkamp, Emily; Giersch, Rachael M.; Wedeman, Emily
Frontiers in Microbiology, Vol. 8
DOI: 10.3389/fmicb.2017.01773

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals
journal, November 2017

Shi, Shuobo; Zhao, Huimin
Frontiers in Microbiology, Vol. 8
DOI: 10.3389/fmicb.2017.02185

Terpenoid Metabolic Engineering in Photosynthetic Microorganisms
journal, October 2018

Vavitsas, Konstantinos; Fabris, Michele; Vickers, Claudia
Genes, Vol. 9, Issue 11
DOI: 10.3390/genes9110520

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC
text, January 2018

Eng, Thomas; Demling, Philipp; Herbert, Robin A.
RWTH Aachen University
DOI: 10.18154/rwth-2019-00645

Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
journal, November 2017

Lian, Jiazhang; HamediRad, Mohammad; Hu, Sumeng
Nature Communications, Vol. 8, Issue 1
DOI: 10.1038/s41467-017-01695-x

Designing with living systems in the synthetic yeast project
journal, July 2018

Szymanski, Erika; Calvert, Jane
Nature Communications, Vol. 9, Issue 1
DOI: 10.1038/s41467-018-05332-z

Multiplexed CRISPR technologies for gene editing and transcriptional regulation
journal, March 2020

McCarty, Nicholas S.; Graham, Alicia E.; Studená, Lucie
Nature Communications, Vol. 11, Issue 1
DOI: 10.1038/s41467-020-15053-x

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion
journal, January 2018

Ferreira, Raphael; Teixeira, Paulo Gonçalves; Siewers, Verena
Proceedings of the National Academy of Sciences, Vol. 115, Issue 6
DOI: 10.1073/pnas.1715282115

Multiplex genome editing of microorganisms using CRISPR-Cas
journal, April 2019

Adiego-Pérez, Belén; Randazzo, Paola; Daran, Jean Marc
FEMS Microbiology Letters, Vol. 366, Issue 8
DOI: 10.1093/femsle/fnz086

Design principles for nuclease-deficient CRISPR-based transcriptional regulators
journal, April 2018

Jensen, Michael K.
FEMS Yeast Research, Vol. 18, Issue 4
DOI: 10.1093/femsyr/foy039

A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast
journal, November 2018

Hofmann, Anja; Falk, Johannes; Prangemeier, Tim
Nucleic Acids Research, Vol. 47, Issue 1
DOI: 10.1093/nar/gky1191

Gene repression via multiplex gRNA strategy in Y. lipolytica
journal, April 2018

Zhang, Jin-lai; Peng, Yang-Zi; Liu, Duo
Microbial Cell Factories, Vol. 17, Issue 1
DOI: 10.1186/s12934-018-0909-8

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC
journal, October 2018

Eng, Thomas; Demling, Philipp; Herbert, Robin A.
Microbial Cell Factories, Vol. 17, Issue 1
DOI: 10.1186/s12934-018-1006-8

Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha
journal, July 2018

Kurylenko, Olena O.; Ruchala, Justyna; Vasylyshyn, Roksolana V.
Biotechnology for Biofuels, Vol. 11, Issue 1
DOI: 10.1186/s13068-018-1203-z

Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production
journal, October 2018

Martins-Santana, Leonardo; Nora, Luisa C.; Sanches-Medeiros, Ananda
Frontiers in Bioengineering and Biotechnology, Vol. 6
DOI: 10.3389/fbioe.2018.00117

Development and Application of CRISPR/Cas in Microbial Biotechnology
journal, June 2020

Ding, Wentao; Zhang, Yang; Shi, Shuobo
Frontiers in Bioengineering and Biotechnology, Vol. 8
DOI: 10.3389/fbioe.2020.00711

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals
journal, November 2017

Shi, Shuobo; Zhao, Huimin
Frontiers in Microbiology, Vol. 8
DOI: 10.3389/fmicb.2017.02185

Terpenoid Metabolic Engineering in Photosynthetic Microorganisms
journal, October 2018

Vavitsas, Konstantinos; Fabris, Michele; Vickers, Claudia
Genes, Vol. 9, Issue 11
DOI: 10.3390/genes9110520

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