Available Technology
In vivo selection systems for evolving the specificity of endonucleases and inteins
Technology:
Plasmid-based selection system and materials
Markets Addressed
Laboratory directed evolution of enzymes with tailor-made DNA cleavage specificities can provide new tools for the site-specific manipulation of genomes for both basic research and gene therapy applications. Since homing endonucleases (HE) are highly specific in their recognition and cleavage of long DNA sequences (14-40 bp), they may be ideally suited for these purposes. However, as there are relatively few HEs available, a method for creating HEs with altered specificities would be important in broadening the potential applicability of these enzymes to gene manipulation and therapy.
Innovations and Advantages
Harvard researchers have developed an efficient in vivo directed evolution system for the selection and assaying of HEs with altered DNA cleavage specificities. One embodiment of the strategy utilizes a pair of matched plasmids to link desired HE catalytic activity to the survival of the E. coli cell harboring the HE library member gene. One plasmid contains a cleavage site of interest together with a caged toxic gene, while the second plasmid encodes the candidate HE. Only cells in which the HE is successful in cleaving the target site can survive (linearization of the plasmid with the toxic gene results in its rapid degradation). The system may utilize both positive and negative selection pressures, either to favor the cleavage of a desired target sequence or to disfavor the cleavage of non-target sequences. The researchers used this system to successfully evolve mutant I-Scel HEs with altered DNA cleave specificities.
This two-plasmid in vivo selection system is highly efficient, easy to use, involves simple plasmid transformation and cell plating rather than requiring protein overexpression, purification, and the performance of in vitro cleavage assays, and should be applicable to other kinds of endonucleases and inteins. In addition, in vivo selection allows enzyme activities to be assayed in the living cell under complex conditions that in some cases may be more relevant than artificial in vitro conditions. The related patent and patent applications describe additional selection systems.
Example two-plasmid selection system #1. The pBar2-sites plasmid contains nuclease cleavage sites of interest and places expression of an amber nonsense mutated barnase gene under control of the arabinose-induced and glucose-repressed pBAD promoter. The pSupE-nuclease plasmid expresses the HE enzyme together with an amber suppressor tRNA.
Example two-plasmid selection system #2. pEndoSce is a low-copy plasmid that expresses I-SceI or a library of I-SceI variants under control of the araBAD promoter. pCcdB is a high-copy plasmid containing multiple copies of the desired or undesired recognition sequence distributed between two locations. This plasmid also expresses the toxic protein CcdB under control of the lac promoter. In the presence of IPTG, nucleases capable of cleaving pCcdB enable cells to survive (positive selection). pCcdB also expresses chloramphenicol acetyltransferase (Cat). In the presence of chloramphenicol and in the absence of IPTG, nucleases capable of cleaving pCcdB induce cell death (negative selection).
Additional Information
Intellectual Property Status: Issued U.S. patent nos.: 7,476,500
Publications:
An In Vivo Selection System for Homing Endonuclease Activity , Gruen, M; Chang, K; Serbanescu, I; Liu, DR. Nucleic Acid Research 30, e29 (2002); PMID: 11917035
Directed evolution and substrate specificity profile of homing endonuclease I-SceI , Doyon, JB; Pattanayak, V; Meyer, CB; Liu, DR. J. Am. Chem. Soc. 128, 2477-2484 (2006); PMID:16478204
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Inventor(s):
Doyon, Jeffrey
Gartner, Zev J.
Gruen, Mathias
Liu, David R.
Rosenbaum, Daniel M.
Categories:
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Vivian Berlin, Director of Business Development
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Reference Harvard Case #2025