Development And Application Of CRISPR-based Combinatorial Regulation

With vast achievements of metabolic engineering and bioproduction,it attracts more and more attention to develop tools for rapid balancing cellular pathways and optimize target productions.Here,we optimized the lightning gRNA-tRNA-array CRISPR-Cas9(GTR-CRISPR)system we developed,which can simultaneously knockout 6 genes of Saccharomyces cerevisiae within 3 days with the knockout efficiency of 63%.In order to combinatorically regulate multiple genes in the mevalonate pathway of Saccharomyces cerevisiae,we optimized the expression of the gRNA-tRNA array through the characterizaiton of different tRNAs,enlarged the targeting range of CRISPR thorugh the application of the dCas9NG protein,and optimized the workflow of combinatorial multiplexed genome regulation through the optimization of the Lightning GTR-CRISPR system,and achieved combinatorial activation of 6 genes in the mevalonate pathway of?-carotene-producing Saccharomyces cerevisiae to different degrees,and obtained a combination that can increase the ?-carotene production of the strain to 4-fold.The combinatorial regulation tool we developed provides a valuable addition to the toolbox of synthetic biology and metabolic engineering.