Fig. 7.4.

Traditional (a) and modern (genome editing, b) methods for construction of improved host strains. (a) Traditional methods can be based on the use of homologous recombination using upstream and downstream sequences (“HR”) of the target gene to be deleted (“Target 1”, “Target 2”) to flank a selective marker (“marker”) as indicated. For each target gene, two consecutive steps (1 and 2) are required for integration of the target site (1) and loop out of the marker (2) before a new target gene can be modified using the same selection (3 and 4). (b) Using CRISPR (illustrated with use of the most used Cas9 nuclease, green box), it is feasible to target modification of two or more genes in a single step using gRNAs directed to each target and a plasmid containing suitable expression cassettes for the Cas9 gene and the gRNAs. If the Cas9 expression cassette is introduced on a plasmid containing, e.g., pyrG as the selective marker, subsequent growth of the resulting strains in medium with FOA results in plasmid (Cas9) loss and enables identification of the desired strains. In the depicted example, two target genes are modified in a single step