Establishing The Methods Of Genome Editing Using CRISPR-Cas9 And Regulating Gene Expression Using Xylose In Trichoderma Reesei

Trichoderma reesei is the most important industrial filamentous fungus for cellulase production and an ideal host for expression of various foreign proteins.However,the current genome editing technology is not mature,and its expression relies heavily on the CBH1 expression system.In this paper,genome editing in T.reesei was studied,and the complementary expression system of CBH1 was developed.The main results of the present study are as follows:1.Preliminary establishment of CRISPR-Cas9 technology in T.reeseiWe tested two gene disruption methods and one method of gene targeted integration in T.reesei using CRISPR-Cas9 in this study.Firstly,the intracellularly expressed Cas9 and transformation of gRNA transcribed in vitro led to unexpected off-target gene disruption in T.reesei QM9414,favoring inserting 9-or 12-bp at 70-and 100-bp downstream of the targeted ura5.Secondly,an alternative method was,established by assembling Cas9 and gRNA in vitro,followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T.reesei TU-6.When the gRNA targeting cbh1 was used,eight among the twenty seven transformants were found to lose the ability to express CBH1,indicative of successful cbh1 disruption through genome editing.Large DNA fragments including the co-transformed plasmid,chromosomal genes,or a mixture of these nucleotides,were inserted precisely in the disrupted cbh1 locus.In addition,this method may also be used for HDR-mediated gene replacement.In a preliminary experiment we have observed gene replacement at the hypothesized ?-glucosidase cel3 c locus,albeit at a low frequency(5 out of 143 transformants),by co-transformation of Cas9-gRNA(targeting cel3c)with a 11.7-kb donor DNA in T.reesei SUS1 strain.2.Xylose-regulated gene expression in T.reeseiTranscriptional factor-based biosensor technology has developed into a detectable signal that reflects the level of intracellular protein expression from the dynamic level,while xylose,as a monomer of xylan,the main component of lignocellulose,has not been widely used in organisms.In this study,the XylR repressor derived from Bacillus subtilis and its own gene modified promoter 77481 were used to establish the biosensor which tested the fluorescent protein DsRed as the reporter gene.It showed an obvious dose-response curve and significantly improved the relative transcription level of DsRed.What's more,in addition to xylose,other sugars such as glucose and mannose can be combined with repressor.Thus we built the recombined expression strain xylR-man5 a,compared with the strain controled with the cbh1 promoter,both were induced in different carbon source.The former expressed relatively high mannase activity.This study provides a complementary strategy to improve the expression of target proteins by significantly utilizing various carbon source inducers generated from lignocellulose degradation.Direct transformation of Cas9-gRNA complex loaded in vitro into the cell is a fast means to disrupt a gene in T.reesei and may find wide applications in strain improvement and functional genomics study.Meanwhile,a biosensor based on bacterial transcription factors was constructed in T.reesei.The binding of sugar inducer to repressor protein changed conformation and could not bind to promoter.According to this feature,xylose was designed to induce the expression of exogenous genes,which expanded the application of lignocellulose degradation products in the induction of T.reesei.