| Candida glycerinogenes is an industrial diploid yeast with excellent multi-stress tolerance,which has been regarded as a potential chassis cell.Nevertheless,the diploid genome,the absence of meiosis and the lack of selectable markers limit the genome editing efficiency of C.glycerinogenes.As a simple,fast and efficient technology,the newly developed CRISPR-Cas9system can easily realize precise genome editing such as point mutation,gene knock-out and knock-in.Thus,it has been widely applied in multiple organisms ranging from microorganisms to plants and animals.In this study,CRISPR-Cas9 based genome editing system was established in C.glycerinogenes,which was then further optimized and applied to the editing of genes such as TRP1,URA3 and ADE2.The principal results are as follows:?i?To establish a CRISPR-Cas9 system in C.glycerinogenes,a yeast integration expression vector pMY that can assemble CRISPR elements such as Cas9 and sgRNA was constructed.Next,the expression levels of different codon-optimized Cas9 genes in C.glycerinogenes were detected by Western Blotting,the result of which confirmed that CgCas9gene was suitable for C.glycerinogenes.Then,the sgRNA transcription intensity of three different promoters?ScSNR52p,GDPp and GAPp?in C.glycerinogenes was tested by targeting the counterselectable marker gene TRP1.The results showed that only the GAP promoter cooperated with HH and HDV ribozyme sequences can realize the knock-out of TRP1 gene with the selective screening efficiency of 100%.?ii?To solve the problems of difficult vector construction and low integration efficiency in the above system,CgCas9 and sgRNA transient expression cassettes were used to replace the CgCas9-sgRNA integrated expression vector.The knock-out of TRP1 gene was also realized with the efficiency of 100%by this simpler CRISPR-Cas9 transient expression system.The 5S rRNA promoter of C.glycerinogenes was identified and tested for TRP1 gene knock-out with the mutation frequency of 3.6×10-6.The result showed that the use of the 5S rRNA promoter instead of the GAP promoter provided a rapid and cloning-free construction method of the sgRNA cassette.To simplify the construction of the repair template,the effect of different length homology arms from 30 bp to 1000 bp on the mutation frequency of TRP1 gene was investigated.The result showed that 50 bp homology arms was the best choice for optimizing the preparation of the repair template.?iii?In order to assess the performance of the optimized transient CRISPR-Cas9 system of C.glycerinogenes,the following applications were carried out:i)the simultaneous knock-out of TRP1 and URA3 genes was achieved with the screening efficiency of 100%and the mutation frequency of 8.7×10-7;ii)by using the recyclable marker gene TRP1,the sequential knock-outs of ADE2 and TRP1 were achieved with the screening efficiency of 80%and 100%respectively;iii)the knock-in of green fluorescent protein?gfp?gene with the efficiency of 100%was realized by adding the coding sequence of gfp gene in the middle of homologous arms of the repair template for TRP1 gene knock-out;iv)xylose dehydrogenase?xylB?gene was inserted into the TRP1 gene locus with the efficiency of 100%,and a xylonic acid producing strain was obtained which produced up to 28.4 g·L-11 ethanol and 9.1 g·L-1 xylonic acid from simulated lignocellulosic hydrolysate(containing 70 g·L-1 glucose and 24 g·L-1 xylose)by two-step fermentation of the dissolved oxygen strategy.These results indicated that the CRISPR-Cas9system developed in this study had a great practical application in the genome editing of C.glycerinogenes. |
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