In vivo modification of a maize engineered minichromosome

Abstract

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

Circular permutation of a synthetic eukaryotic chromosome with the telomerator

“…The linearization function of the telomerator can be achieved with a variety of homing endonucleases (40), CRISPR/Cas systems (41), zinc finger nucleases (42), or TALENs (43) in addition to I-SceI demonstrated here. To build plant artificial chromosomes, endonuclease-dependent linearization of a circular plant artificial chromosome encoding convergent telomere-like sequences has been attempted in vitro before bombardment into maize immature embryos (44); however, it is unclear whether the resulting minichromosomes arose from telomere truncation (45). For in vivo linearization, one important consideration is that the recognition sequence of the enzyme must be sufficiently rare within the sequence of the host genome (i.e., no or few existing sites) to work without causing "collateral damage" in the rest of the genome.…”

Engineered Minichromosomes in Plants

“…Harrington et al (1997) demonstrated this method through formation of de novo minichromosomes by transfecting mammalian cells with alpha satellite arrays, genomic sequences, and human telomere repeats (TTAGGG). Transfection of centromeric sequences into plant systems has been carried out in a number of experiments (Ananiev et al, 2009;Carlson et al, 2007;Phan et al, 2006); however, it is unlikely that de novo centromeric formation has occurred as described previously (Gaeta et al, 2013). The top-down method is a utilization of endogenous chromosomal material through telomere-induced truncation events.…”

“…Such truncated chromosomes are usually not heritable due to possible disruption of genes vital for proper pollen development (Yu et al, 2006). Translocations of acentric fragments and spontaneous tetraploid formation compensate for the loss of these genes and have led to the recovery of A-derived minichromosomes (Gaeta et al, 2013). B chromosomes are supernumerary chromosomes that contain no vital genes, and at low levels, are not deleterious to the containing organism (Carlson and Phillips, 1986).…”

“…It has been recently demonstrated that the inclusion of site-specific recombination cassettes enable in vivo modifications of minichromosomes (Gaeta et al, 2013). This type of manipulation opens the door for possible manipulation strategies, such as gene stacking, to subsequently amend transgene combinations in a targeted manner.…”

Minichromosomes: Vectors for Crop Improvement

“…If an A chromosome is truncated, this will generally lead to a detrimental monosomic condition, though rare events of minichromosomes with an A chromosome centromere and otherwise normal complement of chromosomes have been reported [36]. Truncating a B chromosome has no detrimental effect on the phenotype of a plant and therefore does not lower the rate of recovery of truncation events.…”