CRISPR/Cas9-mediated gene editing vector system for denigration mold.

Heat-resistant filament mold is an industrial silky fungus capable of rapidly degrading cellulose and high-yielding cellulose enzymes, which secrete a wide variety and quantity of hemoctoplasmic cellulose hydrolyzed enzymes, and good high temperature stability, so the bacteria in cellulase production and bio-based fuel research and development has great potential.
currently less research on genetic modification techniques for the heat-destroying mold itself, which greatly limits the application of the industrial fungus.
CRISPR/Cas9 system has been successfully used as an emerging genome editing technology in a range of species and is a new milestone in the study of genomic target modification technology, but the development and use of this technology in thermophilic fungi has not been reported.
Therefore, the development of the technology system for the editing of heat-thyrobacterial mold CRISPR/Cas9 can not only greatly promote the study of the genetic function of the heat-addicted fungus, but also be of great importance to the metabolic transformation of the theophilus metabolism, fermentation to produce biofuels and bio-based chemicals.
Figure 1 CRISPR/Cas9-mediated silk-destroying mold gene editing vector system The microbial functional genomics research team led by Tian Chaoguang, researcher of Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, built a wire-destroying mold genome editing system based on CRISPR/Cas9, using the self-excavated thermally destructive mold RNAase polymer III U6 initiator, which activates the corresponding gRNA in the body. Transcriptional expression, using protosome co-transformation method to the editing system and homogeneity arm into the heat-destroying mold (Figure 1), not only is the same source recombination efficiency is high, but also can simultaneously edit the multi-gene site, of which the dual gene missing isogen recombination efficiency of 61-69%, the homogeneity recombination efficiency of three genes is 30%, the homogeneity recombination efficiency of four genes is 21%.
Figure 2 Multi-gene editing strain cellulase protein secretion relative level Using the system for heat-destroying filamentocycinase secretion pathway for multi-gene editing, you can obtain cellulase protein secretion increased by 5 times the engineering strain (Figure 2).
the technology system is also versatile in other heat-philing fungi, such as isosobacteria.
the research has been funded by scientific and technological programs such as the National Natural Science Foundation of China, and the results have been patented in China, and the results were published in the international journal Biotechnology for Biofuels, with Liu Wei, an associate researcher at Tianjin Institute of Technology, as the first author of the paper.
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