Gene targeting and transgene stacking using intra genomic homologous recombination in plants

Abstract

Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

“…Other challenges including plant species recalcitrance to tissue culture and transformation, and thus low frequency of stably transformed events limit efforts to use genome editing for genetic improvement in some crop species and genotypes ( Figure 3 ) (Shrawat and Lorz, 2006; Hiei et al, 2014; for review, please see Altpeter et al, 2016). Moreover, unintended random integration of donor template should be identified, suppressed or segregated because of biosafety concerns in application of these edited crop plants in a breeding practice ( Figure 3 ) (Altpeter et al, 2016; Kumar et al, 2016). Consequently, substantial efforts have been devoted in the last few years to meet the above challenges.…”

“…Thus, it would be interesting to investigate the effects of different delivery methods and parameters on GT in crop plants. Furthermore, to overcome the challenges of inefficient transformation and plant regeneration systems in a majority of crop species or genotypes, intra-genomic homologous recombination through intra-genomic mobilization by crossing the SSNs transgenic lines with the transgenic plants harboring stable integrated donor DNA template, is an alternative effective strategy not only for GT, but also for gene stacking (Kumar et al, 2016). In addition, taking advantage of geminivirus replicon, a GT enhancement of greater than two orders of magnitude has been achieved (Baltes et al, 2014).…”

“…Virus replicon-based GT could also be a good choice to provide sufficient donor template (Baltes et al, 2014; Cermak et al, 2015). For crop species recalcitrant to transformation and regeneration, intra-chromosome HDR will be an effective alternative strategy (Kumar et al, 2016). Furthermore, suppression of core components of the NHEJ pathway or enhancement of key elements of HDR machinery can be used to increase frequencies of GT (Puchta et al, 1996; Qi et al, 2013; Puchta and Fauser, 2015).…”

“…The presence of a donor DNA containing sequences homologous to those flanking the DSB site can greatly increase the chance of a precise DSB repair through the homology-directed repair (HDR) pathway, leading to gene replacement or foreign gene cassette knock-in as intended (Puchta, 2005; Puchta and Fauser, 2014; Voytas and Gao, 2014; Baltes and Voytas, 2015). Whereas complete knock-outs and loss-of-function mutations are very valuable in defining gene functions, deciphering complex metabolite pathways and becoming routine in plants (Lawrenson et al, 2015; Ma et al, 2015), their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by the random point mutations or indels at specific loci in either the genes’ encoding or promoter regions, change of gene expression levels or insertion of a new gene (Kumar et al, 2016; Sun et al, 2016). So far, target gene replacement or gene targeting (GT) has not yet been well established as a feasible technique in higher plants.…”

“…As a result, the frequency of replacement or targeted integration via HDR is much lower in comparison to random integration (Symington and Gautier, 2011), and reports describing successful gene replacement or site-specific trait gene integration through SSNs-mediated HDR in plants are very limited. Several reviews have described the rapid development and applications of the SSNs system in plants (Voytas and Gao, 2014; Belhaj et al, 2015; Osakabe and Osakabe, 2015; Kumar et al, 2016; Ma et al, 2016; Schiml and Puchta, 2016; Weeks et al, 2016). Here, we present an overview of the recent research advances mainly focusing on development and applications of precise GT in plants using the three recently developed SSN systems, ZFNs, TALENs, and CRISPR/Cas9 reagents, and the potential mechanisms underlying HDR events in plant cells as well as the challenges and future perspectives in implementing precise genome modification through SSNs-mediated GT for crop improvement.…”