title: Development of a genome-editing CRISPR/Cas9 system in thermophilic fungalMyceliophthoraspecies and its application to hyper-cellulase production strain engineering.
abstract: BACKGROUND
  Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi.Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation ofMyceliophthorais crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation ofM. thermophilain biotechnology.
  
  
  RESULTS
  In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic speciesM. thermophilaandM. heterothallica. This CRISPR/Cas9 system could efficiently mutate the importedamdSgene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, includingcre-1,res-1,gh1-1,andalp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain.
  
  
  CONCLUSIONS
  A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification toM. heterothallicaindicates it has wide adaptability and flexibility for use in otherMyceliophthoraspecies. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Ranran
authors.initials: R
authors.lastname: Gao
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Jingen
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Liangcai
authors.initials: L
authors.lastname: Lin
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Wenliang
authors.initials: W
authors.lastname: Sun
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China.
authors.forename: Chaoguang
authors.identifier: 0000-0002-0305-2178
authors.initials: C
authors.lastname: Tian
keywords: CRISPR/Cas9 system; Cellulases; Myceliophthora; Thermophilic fungi