Construction And Application Of A CRISPR-dCas9 System In Candida Tropicalis Based On A TRNA-gRNA Strategy

Candida tropicalis is a non-modal diploid yeast,which has been used as microbial cell factories in producing long-chain dicarboxylic acids,xylitol,hydrogen,bioethanol,etc.As in multi-gene editing experiments in C.tropicalis,gRNA expression was inefficient,endogenous RNA polymerase type Ⅲ promoters urgently need to be explored in efficient guide RNA（gRNA）constructions.In addition,the lack of efficient gene regulation methods limits the development of C.tropicalis metabolic engineering.In this research,a tRNA-gRNA strategy was first created in C.tropicalis.Based on it,a CRISPR-dCas9 gene regulation system was developed.The main results are as follows:（1）Exploration of endogenous tRNA coding genes.The genome sequence of C.tropicalis ATCC 20336 was analyzed by bioinformatics tool tRNAscan-SE,and the coding sequences of tRNAs were obtained.A 71-bp RNA^Glysequence was selected and compared with reported heterogenous tRNA^Gly sequences.The secondary structure of tRNA^Glywas further analyzed,and the potential cleavage sites of cellular nucleases RNase P and RNase Z were speculated.（2）Functional identification of RNA polymerase type Ⅲ promoter and development of a tRNA-gRNA strategy.In CRISPR-Cas9 system,the tRNA^Gly promoter was used for expressing gRNA targeting orotidine-5′-phosphate decarboxylase gene（URA3）and the editing rate was 100%.Utilizing the tRNA^Gly promoter,we designed a tRNA-gRNA strategy to produce multiple gRNAs from a single polycistronic gene.In green fluorescent protein gene（GFP3）,URA3 double-gene knockout experiment,the editing efficiency reached 71.0±4.8%.Additionally,a CRISPR-dCas9 system was constructed in C.tropicalis.（3）Expression regulation of an exogenous gene utilizing CRISPR-dCas9 system.gRNAs were designed to target different regions in coding sequence of GFP3.The transcription level was down regulated to 23.9±4.1%.In addition,detected by flow cytometry,the mean fluorescence intensity of single yeast cell decreased to 34.3±0.5%.Regulating the expression of an endogenous gene utilizing CRISPR-dCas9 system.gRNAs were designed to target different regions of phosphoribosylaminoimidazole carboxylase gene（ADE2）.The transcription level was down regulated to 38.0±7.4%.In C.tropicalis producingβ-carotene,by interfering the expression of squalene synthase gene（ERG9）,β-carotene was increased to2.3 times the amount of the strain without being regulated.