Targeted destruction of intracellular DNA using a CRISPR-based genetic device that can be carried indefinitely in the host genome

Abstract

Environmental release of synthetic DNA resulting from the disposal of spent microbial biocatalyst potentially represents an ecological risk to the environment or a financial risk to biotechnology firms, who might have their intellectual property stolen as a consequence. Thus, a genetically-encoded device that is capable of degrading DNA in a controlled manner would be a valuable and enabling tool. To that end, we have constructed a modular, switchable, genetically-encoded E. coli device for the controlled destruction of user-specified DNA targets in vivo that is based on the organism's native type-IE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) DNA interference (DNAi) pathway. The optimized DNAi device is comprised of two components: a chromosomally-integrated actuator element, which encodes the minimal set of CRISPR-associated (cas) genes required for DNAi activity, and a reprogrammable targeting plasmid, which encodes the CRISPR array specifying the DNA target(s). The device is stable in the OFF state, with >98% of cells retaining a low-copy DNA target over the course of an 8-hr experiment. Upon DNAi activation, the target plasmid is lost from all but 1 in 10⁸ cells and there is a corresponding >10,000-fold decrease in the abundance of the target DNA sequence as recovered by PCR. When the device is targeted to the host genome instead of a plasmid, activation also results in the self-destruction of the host, killing all but -1 in 10⁸ of cells in the ON state but with no appreciable effect on cell viability in the OFF state. Further characterization has also revealed that when DNAi activity is maintained in the OFF state, the overall maintenance cost to the host strain is exceedingly low; the device remains functionally stable over hundreds of cell generations in continuous culture, has little-to-no impact on host growth or plasmid stability, and doesn't interfere with ectopic over-expression of other proteins. The DNAi device is therefore a powerful tool that can potentially be combined with other genetically engineered systems to create safer and more secure forms of biotechnology.