A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable

Leynaud- Kieffer, Laure M. C.; Curran, Samuel C.; Kim, Irene; Magnuson, Jon K.; Gladden, John M.; Baker, Scott E.; Simmons, Blake A.

doi:10.1371/journal.pone.0210243

A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable

Journal Article · Wed Jan 16 23:00:00 EST 2019 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0210243· OSTI ID:1508517

^[1]; Curran, Samuel C. ^[2]; Kim, Irene ^[3]; ^[4]; Gladden, John M. ^[5]; ^[4]; Simmons, Blake A. ^[6]

Swiss Federal Institute of Technology Lausanne (Switzerland); Joint Bioenergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint Bioenergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States)
Joint Bioenergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Joint Bioenergy Institute, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Joint Bioenergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "popout" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Vehicle Technologies Office (EE-3V)

Grant/Contract Number:: AC05-76RL01830; AC02-05CH11231

OSTI ID:: 1508517

Alternate ID(s):: OSTI ID: 1559178

Report Number(s):: PNNL-SA--142504

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 1 Vol. 14; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

References (48)

Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency Jensen, Kristopher Torp; Fløe, Lasse; Petersen, Trine Skov FEBS Letters, Vol. 591, Issue 13 https://doi.org/10.1002/1873-3468.12707	journal	June 2017
A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance Boeke, Jef D.; La Croute, Francois; Fink, Gerald R. Molecular and General Genetics MGG, Vol. 197, Issue 2 https://doi.org/10.1007/bf00330984	journal	November 1984
Biosynthesis of Calcite Nanocrystal by a Novel Polyextremophile Bhargavaea cecembensis-Related Strain Isolated from Sandy Soil Elmi, Fatemeh; Etemadifar, Zahra; Emtiazi, Giti Microbial Ecology, Vol. 85, Issue 2 https://doi.org/10.1007/s00248-022-01977-y	journal	February 2022
Recombinant expression systems in the pharmaceutical industry Schmidt, F. R. Applied Microbiology and Biotechnology, Vol. 65, Issue 4 https://doi.org/10.1007/s00253-004-1656-9	journal	July 2004
Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger Reilly, Morgann C.; Kim, Joonhoon; Lynn, Jed Applied Microbiology and Biotechnology, Vol. 102, Issue 4 https://doi.org/10.1007/s00253-017-8717-3	journal	January 2018
Ku80 Gene is Related to Non-Homologous End-Joining and Genome Stability in Aspergillus niger Zhang, Jinxiang; Mao, Zhihui; Xue, Wei Current Microbiology, Vol. 62, Issue 4 https://doi.org/10.1007/s00284-010-9853-5	journal	January 2011
Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5′-decarboxylase gene, pyrG, as a unique transformation marker d'Enfert, Christophe Current Genetics, Vol. 30, Issue 1 https://doi.org/10.1007/s002940050103	journal	June 1996
Cloning, characterization and molecular docking of a highly thermostable β-1,4-glucosidase from Thermotoga petrophila Haq, Ikram Ul; Khan, Mahmood Ali; Muneer, Bushra Biotechnology Letters, Vol. 34, Issue 9 https://doi.org/10.1007/s10529-012-0953-0	journal	June 2012
Improving cellulase production by Aspergillus niger using adaptive evolution Patyshakuliyeva, Aleksandrina; Arentshorst, Mark; Allijn, Iris E. Biotechnology Letters, Vol. 38, Issue 6 https://doi.org/10.1007/s10529-016-2060-0	journal	February 2016
Improvement of Soybean; A Way Forward Transition from Genetic Engineering to New Plant Breeding Technologies Rahman, Saleem Ur; McCoy, Evan; Raza, Ghulam Molecular Biotechnology, Vol. 65, Issue 2 https://doi.org/10.1007/s12033-022-00456-6	journal	February 2022
Genetic engineering of filamentous fungi — Progress, obstacles and future trends Meyer, Vera Biotechnology Advances, Vol. 26, Issue 2 https://doi.org/10.1016/j.biotechadv.2007.12.001	journal	March 2008
CRISPR system in filamentous fungi: Current achievements and future directions Deng, Huaxiang; Gao, Ruijie; Liao, Xiangru Gene, Vol. 627 https://doi.org/10.1016/j.gene.2017.06.019	journal	September 2017
Highly efficient gene targeting in the Aspergillus niger kusA mutant Meyer, Vera; Arentshorst, Mark; El-Ghezal, Aymen Journal of Biotechnology, Vol. 128, Issue 4 https://doi.org/10.1016/j.jbiotec.2006.12.021	journal	March 2007
Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology Zheng, Yan-Mei; Lin, Fu-Long; Gao, Hao Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-10052-3	journal	August 2017
A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans Chaveroche, M. -K. Nucleic Acids Research, Vol. 28, Issue 22 https://doi.org/10.1093/nar/28.22.e97	journal	November 2000
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems DiCarlo, James E.; Norville, Julie E.; Mali, Prashant Nucleic Acids Research, Vol. 41, Issue 7, p. 4336-4343 https://doi.org/10.1093/nar/gkt135	journal	March 2013
Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation Sugawara, N.; Haber, J. E. Molecular and Cellular Biology, Vol. 12, Issue 2 https://doi.org/10.1128/mcb.12.2.563-575.1992	journal	February 1992
Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells Elliott, Beth; Richardson, Christine; Winderbaum, Jamie Molecular and Cellular Biology, Vol. 18, Issue 1 https://doi.org/10.1128/mcb.18.1.93	journal	January 1998
Development of apyrGMutant ofAspergillus oryzaeStrain S1 as a Host for the Production of Heterologous Proteins Ling, Selina Oh Siew; Storms, Reginald; Zheng, Yun The Scientific World Journal, Vol. 2013 https://doi.org/10.1155/2013/634317	journal	January 2013
Discovery and characterization of ionic liquid-tolerant thermophilic cellulases from a switchgrass-adapted microbial community Gladden, John M.; Park, Joshua I.; Bergmann, Jessica Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-15	journal	January 2014
Methods for genetic transformation of filamentous fungi Li, Dandan; Tang, Yu; Lin, Jun Microbial Cell Factories, Vol. 16, Issue 1 https://doi.org/10.1186/s12934-017-0785-7	journal	October 2017
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR Haeussler, Maximilian; Schönig, Kai; Eckert, Hélène Genome Biology, Vol. 17, Issue 1 https://doi.org/10.1186/s13059-016-1012-2	journal	July 2016
The synergetic effects of two CCAAT boxes in Aspergillus niger glaA gene promoter on activation of PglaA transcription Zhu, Xingguo Science in China Series C, Vol. 47, Issue 2 https://doi.org/10.1360/03yc0097	journal	January 2004
A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi Nødvig, Christina S.; Nielsen, Jakob B.; Kogle, Martin E. PLOS ONE, Vol. 10, Issue 7 https://doi.org/10.1371/journal.pone.0133085	journal	July 2015
Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger Song, Letian; Ouedraogo, Jean-Paul; Kolbusz, Magdalena PLOS ONE, Vol. 13, Issue 8 https://doi.org/10.1371/journal.pone.0202868	journal	August 2018
A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance Boeke, Jef D.; La Croute, Francois; Fink, Gerald R. Molecular and General Genetics MGG, Vol. 197, Issue 2 https://doi.org/10.1007/BF00330984	journal	November 1984
CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art Shi, Tian-Qiong; Liu, Guan-Nan; Ji, Rong-Yu Applied Microbiology and Biotechnology, Vol. 101, Issue 20 https://doi.org/10.1007/s00253-017-8497-9	journal	September 2017
An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency Gems, David; Johnstone, Iain L.; Clutterbuck, A. John Gene, Vol. 98, Issue 1 https://doi.org/10.1016/0378-1119(91)90104-J	journal	February 1991
Filamentous fungi as cell factories for heterologous protein production Punt, Peter J.; van Biezen, Nick; Conesa, Ana Trends in Biotechnology, Vol. 20, Issue 5, p. 200-206 https://doi.org/10.1016/S0167-7799(02)01933-9	journal	May 2002
Production of recombinant proteins by filamentous fungi Ward, Owen P. Biotechnology Advances, Vol. 30, Issue 5 https://doi.org/10.1016/j.biotechadv.2011.09.012	journal	September 2012
Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases Zischewski, Julia; Fischer, Rainer; Bortesi, Luisa Biotechnology Advances, Vol. 35, Issue 1 https://doi.org/10.1016/j.biotechadv.2016.12.003	journal	January 2017
Gene targeting in filamentous fungi: the benefits of impaired repair Krappmann, Sven Fungal Biology Reviews, Vol. 21, Issue 1 https://doi.org/10.1016/j.fbr.2007.02.004	journal	February 2007
Characterization of a polyketide synthase in Aspergillus niger whose product is a precursor for both dihydroxynaphthalene (DHN) melanin and naphtho-γ-pyrone Chiang, Yi-Ming; Meyer, Kristen M.; Praseuth, Michael Fungal Genetics and Biology, Vol. 48, Issue 4 https://doi.org/10.1016/j.fgb.2010.12.001	journal	April 2011
Genome editing in Ustilago maydis using the CRISPR–Cas system Schuster, Mariana; Schweizer, Gabriel; Reissmann, Stefanie Fungal Genetics and Biology, Vol. 89 https://doi.org/10.1016/j.fgb.2015.09.001	journal	April 2016
Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli Nødvig, Christina S.; Hoof, Jakob B.; Kogle, Martin E. Fungal Genetics and Biology, Vol. 115 https://doi.org/10.1016/j.fgb.2018.01.004	journal	June 2018
Food-processing enzymes from recombinant microorganisms—a review Olempska-Beer, Zofia S.; Merker, Robert I.; Ditto, Mary D. Regulatory Toxicology and Pharmacology, Vol. 45, Issue 2 https://doi.org/10.1016/j.yrtph.2006.05.001	journal	July 2006
Cas9 as a versatile tool for engineering biology Mali, Prashant; Esvelt, Kevin M.; Church, George M. Nature Methods, Vol. 10, Issue 10 https://doi.org/10.1038/nmeth.2649	journal	September 2013
Strategies for the transformation of filamentous fungi Ruiz-Diez, B. Journal of Applied Microbiology, Vol. 92, Issue 2 https://doi.org/10.1046/j.1365-2672.2002.01516.x	journal	February 2002
Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Rouet, P.; Smih, F.; Jasin, M. Proceedings of the National Academy of Sciences, Vol. 91, Issue 13 https://doi.org/10.1073/pnas.91.13.6064	journal	June 1994
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Jinek, M.; Chylinski, K.; Fonfara, I. Science, Vol. 337, Issue 6096, p. 816-821 https://doi.org/10.1126/science.1225829	journal	June 2012
The new frontier of genome engineering with CRISPR-Cas9 Doudna, Jennifer A.; Charpentier, Emmanuelle Science, Vol. 346, Issue 6213 https://doi.org/10.1126/science.1258096	journal	November 2014
Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation. Sugawara, N.; Haber, J. E. Molecular and Cellular Biology, Vol. 12, Issue 2 https://doi.org/10.1128/MCB.12.2.563	journal	February 1992
Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells Elliott, Beth; Richardson, Christine; Winderbaum, Jamie Molecular and Cellular Biology, Vol. 18, Issue 1 https://doi.org/10.1128/MCB.18.1.93	journal	January 1998
Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases Bibikova, M.; Carroll, D.; Segal, D. J. Molecular and Cellular Biology, Vol. 21, Issue 1 https://doi.org/10.1128/MCB.21.1.289-297.2001	journal	January 2001
Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases Zischewski, Julia; Fischer, Rainer; Bortesi, Luisa RWTH Aachen University https://doi.org/10.18154/rwth-2017-01660	text	January 2017
Microbial Enzymes: Tools for Biotechnological Processes Adrio, Jose; Demain, Arnold Biomolecules, Vol. 4, Issue 1 https://doi.org/10.3390/biom4010117	journal	January 2014
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR Haeussler, Maximilian; Schönig, Kai; Eckert, Hélène Figshare https://doi.org/10.6084/m9.figshare.c.3643583	collection	January 2016
Nucleosomes impede Cas9 access to DNA in vivo and in vitro Horlbeck, Max A.; Witkowsky, Lea B.; Guglielmi, Benjamin eLife, Vol. 5 https://doi.org/10.7554/eLife.12677	journal	March 2016

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