Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering.

Natalia Rosalía Morero,Sachi Okamoto,Cecilia Zuliani,Fred Dyda,Michael Chandler,Banushree Kumar,Catherine Guynet,Alison Burgess Hickman,Orsolya Barabas,Aleksandra Bebel

doi:10.1093/nar/gky235

Abstract

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction network between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions.

Highlights

Transposable elements (TEs) are a large, ubiquitous group of mobile genetic elements that can autonomously move from one genomic location to another
The complete set of interactions involved in target DNA recognition at LE remained unclear, as the 3 flank of the IPL stem loop, which was predicted to participate in base triplet interactions with GL and target nucleotides [15], was not present in the previous structures
The choice of C for this position was based on the observation that most IS608 integration sites observed in vivo in E. coli contain a C in position +1 after cleavage site (CT) [12]

Summary

Introduction

Transposable elements (TEs) are a large, ubiquitous group of mobile genetic elements that can autonomously move from one genomic location to another. They have had a dynamic role in genome remodelling and evolution, and most eukaryotic and prokaryotic genomes are rich in TE-related sequences [1,2,3,4]. In bacteria TE mobilization has been linked to environmental adaptation and the emergence of multi-drug resistant pathogens [7,8] Due to their inherent ability to carry and integrate DNA into foreign genomes, TEs provide widely used tools for genetic engineering. Much effort has been dedicated to unravel the molecular basis of target DNA selection and transposition of a variety of TEs, in order to optimize TE-based genetic tools and to design strategies to direct their integration to specific genomic sites

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Conclusion