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Nat Commun. 2017 Jan 20;8:14112. doi: 10.1038/ncomms14112.

Rapid generation of hypomorphic mutations.

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Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Department of Biology, Washington University, St Louis, Missouri 63105, USA.
Department of Biology, The University of Western Ontario, 1151 Richmond Street, London, Ontario, Canada N6A5B7.
Science and Technology Branch, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, Canada N5V4T3.
Scattered Gold Biotechnology Inc. 14 Denali Terrace, London, Ontario, Canada N5X 3W2.
Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
The Hope Center for Neurological Diseases, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Department of Genetics, Washington University School of Medicine, St Louis, Missouri 63110, USA.


Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

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