Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

Baxter, S, Lambert, AR, Kuhar, R, Jarjour, J, Kulshina, N, Parmeggiani, F ORCID: https://orcid.org/0000-0001-8548-1090, Danaher, P, Gano, J, Baker, D, Stoddard, B and Scharenberg, A 2012. Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases. Nucleic Acids Research 40 (16) , pp. 7985-8000. 10.1093/nar/gks502

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Abstract

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for similar to 70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy
Publisher:	Oxford University Press
ISSN:	0305-1048
Last Modified:	15 Oct 2024 16:15
URI:	https://orca.cardiff.ac.uk/id/eprint/172207

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