Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease. (5th November 2013)

• Record Type: Journal Article
• Authors: Djukanovic, Vesna; Smith, Jeff; Lowe, Keith; Yang, Meizhu; Gao, Huirong; Jones, Spencer; Nicholson, Michael G.; West, Ande; Lape, Janel; Bidney, Dennis; Carl Falco, Saverio; Jantz, Derek; Alexander Lyznik, Leszek
• Is Part Of: Plant journal. Volume 76:Number 5(2013:Dec.)
• Page Start: 888
• Page End: 899
• Publication Date: 2013-11-05
• Subjects: Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.12335
• Languages: English
• ISSNs: 0960-7412

Abstract

<abstract abstract-type="main" id="tpj12335-abs-0001"> <title>Summary</title> <p>The I–<italic>Cre</italic>I homing endonuclease from <italic>Chlamydomonas reinhardti</italic> has been used as a molecular tool for creating DNA double‐strand breaks and enhancing DNA recombination reactions in maize cells. The DNA‐binding properties of this protein were re‐designed to recognize a 22 bp target sequence in the 5th exon of <italic>MS26</italic>, a maize fertility gene. Three versions of a single‐chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by <italic>Agrobacterium</italic>‐mediated transformation, the cleavage resulted in mutations at a co‐delivered extra‐chromosomal <italic>ms26‐site</italic> in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic <italic>ms26‐site</italic> in 5.8% of transgenic T<sub>0</sub> plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double‐strand break repair generated by non‐homologous end joining. One of 21 mutagenized T<sub>0</sub> plants carried two mutated alleles of the <italic>MS26</italic> gene. As expected, the bi‐allelic mutant T<sub>0</sub> plant and the T<sub>1</sub> progeny homozygous for the <italic>ms26</italic> mutant alleles were male‐sterile. This paper described the second maize chromosomal locus (<italic>liguless‐1</italic> being the first one) mutagenized by a re‐designed I–<italic>Cre</italic>I–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.</p> </abstract>