Design And Application Of CRISPR-mediated Transcriptional Regulatory Signal Amplification Systems In Saccharomyces Cerevisiae

Saccharomyces cerevisiae is an important model eukaryotic microorganism and has been widely applied in fundamental research and the production of various chemicals.Its ability to efficiently and precisely control the expression of multiple genes to key metabolic pathways is valuable for metabolic engineering.The clustered regularly interspaced short palindromic repeats(CRISPR)-mediated regulation enables complex gene expression programming and has been widely used for multi-gene expression regulation.CRISPR activation(CRISPRa)or interference(CRISPRi)can be achieved by fusing nuclease-deficient CRISPR proteins,such as nuclease-deficient Cas9(dCas9),with activation or repression domains of a transcription factor,respectively.The easily programmable single guide RNA(gRNAs)endows this CRISPR-mediated system the ability to target almost to any region of the genome with high efficiency and specificity.The fusion of single transcription effector often cannot achieve efficient transcriptional regulation;thus,to increase the effectiveness of transcriptional regulation,hybrid transcription effectors with several activation or repression domains have been designed.However,the number of proteins for direct fusion remains limited,which restricts the regulation efficiency.On this basis,RNA or protein scaffolds for recruiting multiple transcription effectors have been developed for application in CRISPR regulation,but in S.cerevisiae protein scaffold-mediated CRISPR regulatory systems have not been reported.In addition,only single direction of regulation was reported using protein scaffolds,detailed characterization of protein scaffold-mediated CRISPR system for simultaneous gene repression and activation,which is crucial for multiplexed gene regulation,particularly in S.cerevisiae,is much needed.In this study,we developed an orthogonal CRISPR-mediated protein-tagging signal amplification system for simultaneous multiplexed gene activation and repression for the first time in S.cerevisiae,which achieved efficient amplification of transcriptional regulatory signals.We first screened and optimized transcriptional regulators,followed by introducing two sets of protein scaffolds(SPY and Sun Tag scaffolds)into the CRISPR system,and fused them with nuclease-deficient CRISPR proteins to recruit multiple transcription factors to enhance transcription.In addition,the effect of tandem array of different numbers of peptides on transcriptional regulation was characterized in detail to obtain systems with significantly increased regulatory efficiency,and then orthogonal transcriptional activation and repression regulatory systems were constructed.On this basis,the orthogonal bi-functional CRISPRmediated transcriptional regulation system was used to regulate the expression of genes associated with 3-hydroxypropanoic acid production.The regulation of the enzymes related to malonyl-CoA synthesis improved the production of 3-hydroxypropanoic acid,significantly.This demonstrates that the CRISPR-mediated transcription factor recruitment system is an efficient tool for multiple genes regulation,and its efficiency is better than the current CRISPR regulatory system fused directly with transcription factors.The contents of this study are as follows:(1)Construction and optimization of a dCas9-mediated transcriptional activation signal amplification system.In this study,we first constructed an activation signal amplification system based on dCas9 from Streptococcus pyogenes in S.cerevisiae.Activator strength is the most important factor affecting the activation.We compared the activation efficiency of three different transcriptional activators in yeast,including VP64-p65-Rta(VPR),a heterotrimeric activator protein commonly used in eukaryotic cells;Med2,a yeast endogenous transcriptional activator protein,and dMed2,which only contains the activation domain of Med2.We found that dCas9-Med2 demonstrated the highest activation efficiency;dCas9-Med2 activated the expression is 2.8-fold of the control,which was 29.5%higher than that shown by dCas9-VPR.On this basis,two scaffold proteins,SunTag and SPY,were constructed for the recruitment of multiple transcription activators,and then the effect of scaffold recruitment with different numbers of transcriptional activation proteins on transcriptional efficiency was tested.The activation efficiency of dCas9-10×SunTag-Med2 and dCas9-24×SunTag-Med2 was similar.dCas9-10 × SunTag activated the expression 4.2-fold,with this value being 46%higher than that shown by dCas9-Med2,the direct fusion of transcription factor Med2 with nucleasedeficient CRISPR proteins.Morever,the dCas9-SPY-Med2 system has a higher activation effect than dCas9-SunTag-Med2.We found that the activation efficiency of dCas9-6×SPYMed2 was higher,and can reach 7.9-fold activation.In comparison with dCas9-Med2,the activation efficiency of dCas9-6 × SPY was 3.3-fold higher.These findings demonstrated that both SunTag and SPY amplified the activation efficiency of CRISPR-mediated gene expression and the SPY system was more effective than the SunTag system.In addition,the transcriptional activation of other promoter-controlled GFP expression was tested with the dCas9-SPY-Med2 system.We found that the activation efficiency was enhanced significantly,with the highest activation efficiency reaching to 34.9-fold,which is 3.8-fold higher than that shown by dCas9-Med2.These findings indicated that dCas9-SPY systems exhibited good activation efficiency as well as applicability with different promoters.(2)Construction of dCas9-mediated transcriptional repression signal amplification systemBasis the aforementioned results,we subsequently constructed a CRISPRi signal amplification system.Based on previous reports,the endogenous repressor protein TUP1 of S.cerevisiae was tested as a transcriptional repressor.We observed that dCas9-TUP1 only repressed the expression by 38%.dCas9-24 × SunTag and dCas9-6 × SPY showed higher repression efficiency than dCas9-TUP1.They can repress GFP expression by 73%and 63%,respectively,being 2.2-and 1.6-fold higher than that shown by dCas9-TUP1.These results indicated both the CRISPR-mediated SunTag and SPY systems achieved better repression than dCas9-TUP1,with the SunTag system showing the best repression effect.The dCas9SunTag-TUP1 system also worked effectively with other promoters.(3)Construction and optimization of an efficient orthogonal bifunctional CRISPRmediated transcriptional regulation systemTo develop an orthogonal bifunctional CRISPR-mediated transcriptional regulation system,nuclease-deficient CRISPR proteins,dCpf1 from Lachnospiraceae bacterium ND2006,was introduced to construct a transcriptional repression system to achieve an orthogonal with the dCas9-SPY-Med2 system.However,although the repression efficiency of dCpf1-SunTag-TUP1 was higher than that of dCpfl-TUP1,only 44%repression was achieved.To improve repression efficiency,we evaluated and compared the inhibitory effects of the repression domains of endogenous TUP1,MIG1 and heterologous Mxi1.dCpf1-MIG1 showed a better inhibitory effect,reaching to 50%.We then selected MIG1 as the repression domain to establish a repressor recruiting system.Comparing with dCpf1-MIG 1,dCpf1SunTag showed much higher inhibition of GFP expression,reaching up to 77%.We also examined the repression of GFP expression driven by different promoters.The repression efficiency can reach to 95%,using dCpf1-SunTag-MIG1,being 7.9-higher than that shown by dCpf1-MIG1.These findings demonstrated that highly efficient repression could be achieved by dCpf1-SunTag.We used two systems,dCas9-SPY-Med2 and dCpf1-SunTag-MIG1,to simultaneously up-regulate and down-regulate different fluorescent reporter proteins,respectively,and found that the efficiency of up-regulation or down-regulation was consistent with that of single signal amplification system.This indicated that these two CRISPR-mediated systems were orthogonal,with no crosstalk between them and thus an orthogonal CRISPR-based signal amplification system with dual functions has been successfully developed.(4)Combinatorial regulation of 3-hydroxypropanoic acid production using CRISPRa/i signal amplification systemsTo detect CRISPRa/i orthogonality and application,as a proof-of-concept study,we used CRISPRa/i signal amplification systems to regulate the metabolic pathway for 3hydroxypropanoic acid production.We up-regulate ACC1,a key gene in the metabolic pathway,with the activation system,and down-regulate FAS1,FAS2 and ARG3 with the repression system.The results showed that 3-hydroxypropanoic acid production levels were improved by 31%when dCas9-6 × SPY was used to activate ACC1 expression.Similarly,3HP production level was improved by 55%when dCpfl-SunTag-TUP1 was used to repress FAS1 and FAS2.Compared with only downregulation of FAS1 and FAS2,when simultaneous upregulation of ACC1 and downregulation of FAS1 and FAS2,3-hydroxypropanoic acid production level was further increased by 19%.In addition,combing FAS1,FAS2 and ARG3 down-regulation and ACC1 upregulation,the production of 3-hydroxypropanoic acid was further increased by 16%,demonstrated the advantage of our CRISPR-based system in multiple gene regulation.The results of malonyl coenzyme A level monitored by biosensors were also consistent with the production of 3-hydroxypropionic acid.To summarize,we developed two CRISPR-mediated signal amplification systems in S.cerevisiae to recruit mutiple transcription factors and the system significantly improved transcriptional regulation efficiency by.The design of two CRISPR protein scaffold regulatory systems was used to achieve orthogonal activation and repression signals.This system is a novel CRISPR regulatory system with better gene regulation efficiency than the commonly used CRISPR systems that directly fuse transcription factors.We believe it will be a valuable tool for both metabolic engineering and fundamental studies.