Stable integration of an engineered megabase repeat array into the maize genome

Abstract

Plant genome engineering as a practical matter will require stable introduction of long and complex segments of DNA sequence into plant genomes. Here we show that it is possible to synthetically engineer and introduce centromere-sized satellite repeat arrays into maize. We designed a synthetic repeat monomer of 156 bp that contains five DNA-binding motifs (LacO, TetO, Gal4, LexA, and CENPB), and extended it into tandem arrays using an overlapping PCR method similar to that commonly used in gene synthesis. The PCR products were then directly transformed into maize using biolistic transformation. We identified three resulting insertion sites (arrayed binding sites), the longest of which is at least 1100 kb. The LacI DNA-binding module is sufficient to efficiently tether YFP to the arrayed binding sites. We conclude that synthetic repeats can be delivered into plant cells by omitting passage through Escherichia coli, that they generally insert into one locus, and that great lengths may be achieved. It is anticipated that these experimental approaches will be useful for future applications in artificial chromosome design.