title: Targeted DNA degradation using a CRISPR device stably carried in the host genome.
abstract: Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 10(8)-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10(8). Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months.
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Caliando
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
mesh_terms: D064113:CRISPR-Cas Systems; D016923:Cell Death; D049109:Cell Proliferation; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering; D005821:Genetic Techniques; D016678:Genome; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D004269:DNA, Bacterial; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein