A photoresponsive homing endonuclease for programmed DNA cleavage

Johnson, Luke A. ORCID: https://orcid.org/0000-0002-6697-6589, Mart, Robert J. ORCID: https://orcid.org/0000-0003-2196-5840 and Allemann, Rudolf K. ORCID: https://orcid.org/0000-0002-1323-8830 2024. A photoresponsive homing endonuclease for programmed DNA cleavage. ACS Synthetic Biology 13 (1) , pp. 195-205. 10.1021/acssynbio.3c00425

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Abstract

Homing endonucleases are used in a wide range of biotechnological applications including gene editing, in gene drive systems, and for the modification of DNA structures, arrays, and prodrugs. However, controlling nuclease activity and sequence specificity remain key challenges when developing new tools. Here a photoresponsive homing endonuclease was engineered for optical control of DNA cleavage by partitioning DNA binding and nuclease domains of the monomeric homing endonuclease I-TevI into independent polypeptide chains. Use of the Aureochrome1a light-oxygen-voltage domain delivered control of dimerization with light. Illumination reduced the concentration needed to achieve 50% cleavage of the homing target site by 6-fold when compared to the dark state, resulting in an up to 9-fold difference in final yields between cleavage products. I-TevI nucleases with and without a native I-TevI zinc finger motif displayed different nuclease activity and sequence preference impacting the promiscuity of the nuclease domain. By harnessing an alternative DNA binding domain, target preference was reprogrammed only when the nuclease lacked the I-TevI zinc finger motif. This work establishes a first-generation photoresponsive platform for spatiotemporal activation of DNA cleavage.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Chemistry
Publisher:	American Chemical Society
ISSN:	2161-5063
Funders:	BBSRC
Date of First Compliant Deposit:	11 December 2023
Date of Acceptance:	13 November 2023
Last Modified:	23 Jan 2024 15:07
URI:	https://orca.cardiff.ac.uk/id/eprint/164664

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