Development and application of multiple genome editing method using CRISPR/Cas9 system and ribozyme in Escherichia coli

Abstract

Engineering cellular metabolism for improved production of valuable products requires extensive modulation of bacterial genome to explore complex genetic spaces. In order to introduce genetic modifications for rebalancing the metabolic flux, target genes to be modulated should be determined, and the expression of target genes should be optimized. However, it is difficult to select the target genes precisely because it is very labor-intensive to identify all the effects of the expression changes of the all genes involved in the metabolic pathway on the production of the target product. In addition, even if several genes were selected to be modulated, it is difficult to optimize the metabolic pathway because the combination of modulation may cause detrimental effects on the strain. Therefore, in order to overcome the drawbacks described above, it is reasonable to conduct the metabolic engineering using a combinatorial approach. A combinatorial approach is to screen strain with the best phenotype among the various mutation library expected to improve the desired phenotype. Therefore, in order to generate the mutant library, the tool to introduce mutations at multiple loci is required. <br/> In this thesis, we established the multiple sgRNA generation strategy to enable the metabolic pathway optimization using the CRISPR/Cas system. The dCas9-ω, which fused the transcriptional activator domain to inactivated cas9 (dCas9), can activate or repress target gene expression depending on the design of the sgRNA. Therefore, if multiple sgRNAs can be generated, the expression of several genes can be modulated simultaneously without genetic modification. For this reason, the strategy to generate multiple sgRNAs was required, and we constructed a strategy to produce several sgRNAs from one primary transcript using the self-cleavage property of Rz. By combining multiple sgRNA production strategies with the dCas9-ω system, target genes that have a strong effect on phenotypic enhancement can be efficiently identified.<br/> After identification of target genes to be modulated, genetic engineering should be performed to alter the expression of those genes. Modulating in the expression level of a single gene to fine tune the metabolic flux often results in a change in the overall flux. Therefore, when optimizing the expression levels of target genes to achieve the desired phenotype through the metabolic engineering, the combinatorial approach is more reasonable than the sequential approach. To do this, it is necessary to be able to construct a library of sufficient size that incorporates genetic modifications to various locations within the chromosome. However, the multiplexing methods reported in the literature have limitations in producing mutant libraries for applying the combinatorial approach because the recombination efficiency is very low and negative selection is not efficient. Therefore, we have designed a system that allows a combinatorial approach by using the CRISPR/Cas system to introduce a mutation library for one site in one cycle and accumulate a mutation library for multiple sites by repeating this cycle. Therefore, we have constructed a plasmid capable of obtaining high CFU with high editing efficiency through the CRISPR/Cas system, and confirmed that a strain in which mutations were introduced into three different target genes could be generated with high efficiency.<br/>