Combinational Repression Of Multi-gene Based On CRISPRi

Optimizing metabolic flux by regulating the expression of key enzymes is one of the important ways to increase the titer and yield of metabolic engineering products.There are usually hundreds to thousands of chemical reactions in microbial cell factories,which interact to form a large and densely connected network.Therefore,the combined regulation of multi-gene is necessary.There are a variety of means to achieve this,such as promoter modification,RBS optimization,gene knock-out,riboswitch control,etc.These methods require genetic engineering to transform the genetic information of the cell factory,which is tedious and difficult to operate.Alternatively,some other methods that do not require gene recombination.Hfq-dependent small RNA,antisense RNA(asRNA)and RNAi hinder the translation of mRNA into protein to regulate gene expression.However,this method has obvious off-target and cytotoxicity.Recently,CRISPRi has attracted much attention in the field of gene regulation.With only the catalytically inactivated Cas protein and a chimeric single-guide RNA(sgRNA),CRISPRi can repress gene expression through inhibiting transcription and exhibit high inhibition efficiency and specificity in bacteria.About metabolic engineering,this simple and low-cost tool has been established in more and more model strains and non-model strains to remold metabolic pathways,identify new highly efficient metabolic enzymes,and optimize chassis cells.There are already many applications that use CRISPRi to simultaneously regulate multiple metabolic reactions,most of them have achieved higher titers than single-gene repression.However,these applications are still at a relatively preliminary stage,which expresses multiple gRNAs through several constitutive promoters or the same inducible promoter to achieve simultaneous regulation of multi-genes and multiple pathways.There are some limits to this method.First,the expression of each pathway cannot be adjusted independently.In the metabolic network,the regulation of each metabolic pathway has its optimal time and strength.In addition,in order to find the best combination of multi-gene repression,it is necessary to construct a large number of gRNA expression plasmids,which was time-consuming and labor-intensiveThis paper aims to independently regulate the expression of three different gRNAs by three orthogonal and rigorous expression promoters to achieve the combination and timing control of the three genes.The main difficulty of the experiment is that the sensitivity of the CRISPRi system leads to a slight leakage of the promoter that induces gRNA expression,which can lead to the suppression of the expression of the target gene.To solve this problem.we first screened and optimized the promoters that make up the system.Finally,three inducible expression systems with extremely low leakage expression levels were obtained,which were combined into a multi-gene inducible expression system to serve as a gRNA expression platform There are two identical IIS type endonuclease recognition sites downstream of each promoter,and three genes or spacers can be inserted at once by the one-step ligation method based on Golden Gate.After that,three different fluorescent protein reporter genes were used to test the system,indicating that it has the ability of combined expression and sequential expression.The second part is the establishment of multiple gene suppression systems.Insert a gRNA backbone downstream of the three inducible promoter sequences,and insert the gene sequence of dCas9 protein in the TFs expression plasmid to complete the preliminary construction of the multi-gene suppression system.The system achieves the suppression of the expression of multiple genes,but the suppression caused by gRNA leakage is still relatively serious.So we mutated the gRNA backbone to reduce its affinity with the dCas9 protein,which further weakened the gene suppression caused by gRNA leakage expression.Then we explored the factors affecting the inhibitory effect of mutant gRNA from the three aspects of gRNA binding site,spacer length and inducible promoter to achieve the best inhibitory effect.The third part is the application of the system in NeuAc production.In order to show the application value of this system in the field of metabolic engineering,we use it in optimizing NeuAc production.We designed targeting sites for ten genes related to the consumption of NeuAc precursor PEP,to characterize the help of single gene suppression on NeuAc production.Then we selected 4 targets and combined them in groups of three to construct a multi-gene suppression plasmid,which ultimately increased the yield of NeuAc by 1.41-fold.The CRISPRi-based multi-gene combination repression system we constructed can achieve combined and sequential repression of three genes.Because the gRNA combination is replaced with the combination of the inducer,the number of gRNA expression plasmids is reduced,then the workload is reduced,and a lot of time and scientific research resources are saved.At the same time,the degree and time of gene repression can also be flexibly adjusted according to the growth stage of the bacteria to avoid the occurrence of situations such as excessive or premature inhibition of key genes that affect growth,and achieve the optimal regulatory effect.