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

Shur, Andrey and Murray, Richard M. (2017) Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli. . (Submitted)

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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.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper Paper
Murray, Richard M.0000-0002-5785-7481
Additional Information:The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-ND 4.0 International license. bioRxiv preprint first posted online Feb. 21, 2017. AS was supported by the NIH training grant number 5T32GM007616-37. Research supported in part by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.
Funding AgencyGrant Number
NIH Predoctoral Fellowship5T32GM007616-37
Army Research Office (ARO)W911NF-09-0001
Record Number:CaltechAUTHORS:20170627-105346578
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:78610
Deposited By: Tony Diaz
Deposited On:27 Jun 2017 19:30
Last Modified:15 Nov 2021 17:41

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