CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside

Cpf1 enables fast and efficient genome editing in Aspergilli

Additional file 1: Fig. S1. Cloning procedure of tRNA based gRNA expression. a Primer pair sets for amplifying the bipartite gRNA biobricks. b Bipartite gRNA biobricks after PCR amplification. We note that one biobrick (P1 + 2) is constant for all experiments, as only the PCR fragment (Px + 4) containing the protospacer needs to be specifically produced for each new experiment. c Design of the primer tails for USER fusion of bipartite gRNA biobricks. d Cpf1-CRISPR vector fragment (pAC1430) after linearization with PacI and Nt.BbvCI. e Insertion of gRNA biobrick into Cpf1-CRISPR vector (pAC1430) by USER cloning in E. coli