skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

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.

Authors:
ORCiD logo [1];  [2];  [3]; ORCiD logo [4];  [5]; ORCiD logo [4];  [6]
  1. Swiss Federal Institute of Technology Lausanne (Switzerland); Joint Bioenergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Joint Bioenergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  3. Univ. of California, Berkeley, CA (United States)
  4. Joint Bioenergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  5. Joint Bioenergy Institute, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  6. Joint Bioenergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
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)
OSTI Identifier:
1508517
Report Number(s):
PNNL-SA-142504
Journal ID: ISSN 1932-6203
Grant/Contract Number:  
AC05-76RL01830; AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 14; Journal Issue: 1; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Leynaud- Kieffer, Laure M. C., Curran, Samuel C., Kim, Irene, Magnuson, Jon K., Gladden, John M., Baker, Scott E., and Simmons, Blake A. A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable. United States: N. p., 2019. Web. doi:10.1371/journal.pone.0210243.
Leynaud- Kieffer, Laure M. C., Curran, Samuel C., Kim, Irene, Magnuson, Jon K., Gladden, John M., Baker, Scott E., & Simmons, Blake A. A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable. United States. doi:10.1371/journal.pone.0210243.
Leynaud- Kieffer, Laure M. C., Curran, Samuel C., Kim, Irene, Magnuson, Jon K., Gladden, John M., Baker, Scott E., and Simmons, Blake A. Thu . "A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable". United States. doi:10.1371/journal.pone.0210243. https://www.osti.gov/servlets/purl/1508517.
@article{osti_1508517,
title = {A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable},
author = {Leynaud- Kieffer, Laure M. C. and Curran, Samuel C. and Kim, Irene and Magnuson, Jon K. and Gladden, John M. and Baker, Scott E. and Simmons, Blake A.},
abstractNote = {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.},
doi = {10.1371/journal.pone.0210243},
journal = {PLoS ONE},
issn = {1932-6203},
number = 1,
volume = 14,
place = {United States},
year = {2019},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Filamentous fungi as cell factories for heterologous protein production
journal, May 2002


A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
journal, March 2013

  • DiCarlo, James E.; Norville, Julie E.; Mali, Prashant
  • Nucleic Acids Research, Vol. 41, Issue 7, p. 4336-4343
  • DOI: 10.1093/nar/gkt135