Serine Integrases: Advancing Synthetic Biology - ACS Publications

Jan 9, 2018 - sequence and ligation independent cloning. CPEC. circular polymerase extension cloning. SSTRA. site-specific recombination-based tandem ...
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Serine Integrases: Advancing Synthetic Biology Christine Merrick, Jia Zhao, and Susan Rosser ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00308 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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ACS Synthetic Biology

Serine integrases: advancing synthetic biology Authors: Christine A. Merrick1*, Jia Zhao2, Susan J. Rosser1 1.

School of Biological Sciences, University of Edinburgh, Roger Land

Building, Alexander Crum Brown Road, Edinburgh EH9 3FF, UK 2.

Novo Nordisk (China) Pharmaceuticals Co., Ltd., Lei Shing Hong Center,

Guangshunnan Avenue, Beijing 100102, China

*Corresponding author: Email: [email protected] Tel: 0131 650 5416

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Abstract Serine integrases catalyze precise rearrangement of DNA through sitespecific recombination of small sequences of DNA called attachment (att) sites. Unlike other site-specific recombinases, the recombination reaction driven by serine integrases is highly directional and can only be reversed in the presence of an accessory protein called a recombination directionality factor (RDF). The ability to control reaction directionality has led to the development of serine integrases as tools for controlled rearrangement and modification of DNA in synthetic biology, gene therapy, and biotechnology. This review discusses recent advances in serine integrase technologies focussing on their applications in genome engineering, DNA assembly, and logic and data storage devices.

Keywords: serine integrase, recombination directionality factor (RDF), DNA assembly, genome engineering, logic devices, genetic data storage

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ACS Synthetic Biology

DNA manipulation and modification are at the core of synthetic biology. Advances in the capacity to engineer DNA with increasingly complex properties are accelerating a diverse range of medical and biotechnological applications. In recent years, the principles of homologous recombination have been applied to DNA assembly 1 (for example, in construction of the first synthetic genome) 2 and to targeting specific changes in genomic DNA (for example, in nuclease-based genome editing with clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleases like Cas9 and Cpf1) 3-8. However, another group of DNA modifying enzymes – the site-specific recombinases - are becoming established as vital tools for genetic modification both in vivo and in vitro. Unlike homologous recombination, site-specific recombination requires no extensive DNA sequence homology, no synthesis or degradation of DNA and has no reliance on endogenous repair pathways or cofactors. Serine integrases are a subfamily of serine site-specific recombinases, which are evolutionarily and mechanistically distinct from tyrosine site-specific recombinases 9. Serine recombinases make double strand breaks in DNA forming covalent 5’-phosphoserine bonds with the DNA backbone before religation, whereas tyrosine recombinases cleave single strands forming covalent 3’-phosphotyrosine bonds with the DNA backbone and rejoin the strands via a Holliday junction-like intermediate state. In comparison to tyrosine recombinases, recombination by serine integrases has the advantage of simplicity, normally only requiring the integrase protein and small att sites (