Genome-Wide Abolishment of Mobile Genetic Elements Using Genome Shuffling and CRISPR/Cas-Assisted
MAGE Allows the Efficient Stabilization of a Bacterial Chassis.
The ideal bacterial chassis provides a simplified, stable and predictable host environment
for synthetic biological circuits. Mutability and evolution can, however, compromise
stability, leading to deterioration of artificial genetic constructs. By eliminating
certain sources of instability, these undesired genetic changes can be mitigated.
Specifically, deletion of prophages and insertion sequences, nonessential constituents
of bacterial genomes, has been shown to be beneficial in cellular and genetic stabilization.
Here, we sought to establish a rapid methodology to improve the stability of microbial
hosts. The novel workflow involves genome shuffling between a mobile genetic element-free
strain and the target cell, and subsequent rounds of CRISPR/Cas-assisted MAGE on multiplex
targets. The power and speed of the procedure was demonstrated on E. coli BL21(DE3),
a host routinely used for plasmid-based heterologous protein expression. All 9 prophages
and 50 insertion elements were efficiently deleted or inactivated. Together with additional
targeted manipulations (e.g., inactivation of error-prone DNA-polymerases), the changes
resulted in an improved bacterial host with a hybrid (harboring segments of K-12 DNA),
9%-downsized and clean genome. The combined capacity of phage-mediated generalized
transduction and CRISPR/Cas-selected MAGE offers a way for rapid, large scale editing
of bacterial genomes.