Flexible TALEs for an expanded use in gene activation, virulence and scaffold engineering

authored by
Sebastian Becker, Stefanie Mücke, Jan Grau, Jens Boch
Abstract

Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a -1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.

Organisation(s)
Section Plant Biotechnology
Institute of Plant Genetics
External Organisation(s)
Martin Luther University Halle-Wittenberg
Type
Article
Journal
Nucleic Acids Research
Volume
50
Pages
2387-2400
No. of pages
14
ISSN
0301-5610
Publication date
28.02.2022
Publication status
Published
Peer reviewed
Yes
ASJC Scopus subject areas
Genetics
Electronic version(s)
https://doi.org/10.1093/nar/gkac098 (Access: Open)