Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli

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Title Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli
Authors Andrey Shur and Richard M Murray
Source Submitted, 2017 Synthetic Engineering: Engineering, Evolution and Design (SEED) Conference
Abstract Serine integrases are bacteriophage proteins responsible for integrating the phage genome into that of the host. Synthetic biologists have co-opted these proteins into useful tools for permanent DNA logic, utilizing their specific DNA recombination abilities to build synthetic cell differentiation and genetic memory systems. Each integrase has a specific pair of DNA sequences (attP/attB sites) that it recombines, but multiple identical sites can result in unpredictable recombination. We have developed a way to control integrase activity on identical attP/attB sites by using catalytically dead Cas9 (dCas9) as a programmable binding protein that can compete with integrase for binding to specific attachment sites. Utilizing a plasmid that contains two identical Bxb1 attP sites, integration can be repressed up to 8 fold at either one of the two attP sites when guide RNA and dCas9 are present. Guide RNA sequences that bind specifically to attB, or either of two attP sites, have been developed. Future goals are to utilize this technology to construct larger and more complex integrase logic circuits.
Type Conference paper
Tag sm17-seed
ID 2017a
Funding ARO ICB