Establishment And Optimization Of CRISPRi Technology In Yarrowia Lipolytica

The Yarrowia lipolytica,as an oleaginous yeast,has became a promising microbial cell factory bacause of their biochemical characteristics and ability to accumulate lipid-based chemicals.To manipulate metabolic flux and introduce heterogenous biosynthesis pathway into Y.lipolytica,a number of studies have been implemented for developing synthetic biology tools for gene regulation.CRISPR interference?CRISPRi?,as a new technology,has been applied for numerous organisms for repressing genes of interest.In this study,we established CRISPRi systems in Y.lipolytica based on five different repressors,that was dCpf1?DNase-deactivated Cpf1?from Francisella novicida,dCas9?deactivated Cas9?from Streptococcus pyogenes,and three other fusion proteins?dCpf1-KRAB,dCas9-KRAB and dCas9-Mix1?.Based on the GFP reporter system and PVA testing system,we tested whether the CRISPRi systems created in Y.lipolytica could make an effective gene repression and investigated the relationship between targeting site and repression efficiency.Ten gRNAs were designed to bind to different regions of gfp gene,and eight gRNAs binding to different regions of vioE gene were also designed.The regulation results in both systems demonstrated the repression function of CRISPRi system.However,another phenomenon appeared that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used.In order to yield strong gene repression rapidly,we developed a multiplex gRNAs strategy,based on one-step Golden-brick assembly technology.Through making three different gRNAs towards gfp gene simultaneously,high repression efficiency 85%?dCpf1?and92%?dCas9?were achieved in a short time,which avoided the need of screening effective gRNA loci in advance.In addition,we compared the repression efficiency of different synthetic Pol III promoter(SNR52'-tRNA^Glyly and SCR1'-tRNA^Gly)and found that SCR1'-tRNA^Gly performed better in the case of multiple-site repression.Moreover,two genes interference including gfp and vioE and three genes repression including vioA,vioB and vioE in protodeoxy-violaceinic acid?PVA?pathway were also realized.Taken together,based on different repressors of dCpf1,dCas9,dCpf1-KRAB,dCas9-KRAB and dCas9-Mxi1,successful CRISPRi-mediated regulation of gene expression is achieved in Y.lipolytica.And we demonstrate that by using the one-step Golden-brick assembly,the multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene.This timesaving method promised to be a potent transformative tool valuable for metabolic engineering,synthetic biology,and functional genomic studies of Y.lipolytica.