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Background  Thermophilic filamentous fungus  Myceliophthora thermophila  has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR–Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes.    Results  In this study, we developed an  Acidaminococcus  sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in  M. thermophila . These CRISPR–Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR–Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in  M. thermophila  via three transformation rounds, using two selectable markers  neo  and  bar . We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR–Cas9 system and found the two technologies were complementary. Together we called them  CRISPR–Cas - a ssisted  m arker  r ecycling  t echnology (Camr technology).    Conclusions  Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR–Cas12a system or the established CRISPR–Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.

Upgrading of efficient and scalable CRISPR–Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila