Editing Plant Genomes: a new era of crop improvement (2016)

Genome editing comprises predicted changes in the gene sequence or precise insertion of exogenous DNA with the goal of inactivating gene(s), generating functional alleles, replacing mutant alleles or site-specific transgene integration. The first case, representing targeted gene integration, in a plant genome (tobacco) was reported a few years after the birth of the plant transformation era (Paszkowski et al., 1988). However, due to an exceedingly low targeting frequency, this technique could not be practiced routinely. In the meantime, Ow and colleagues (Albert et al., 1995; Dale and Ow, 1991) showed that a phage derived site-specific recombination system, Cre-lox, works exceptionally well in plant cells and directs precise DNA deletions and site-specific transgene integration into plant chromosomes. While targeted gene replacement could not be practiced by employing such heterologous recombination systems, precise transgene integrations into pre-integrated recombination sites and excision of selectable marker genes have since become routine. Around the same time, Puchta et al. (1996) showed how the dilemma of low frequency gene editing in plants could be addressed. By introducing DNA double-stranded breaks (DSB) in a tobacco genome, a dramatic increase in gene integration was demonstrated by homologous recombination (HR), the most powerful gene editing mechanism with regard to controlling the editing outcome. However, the application of the DSB strategy for genome editing had to await the development of designed nucleases. The last decade has seen a number of breakthroughs leading to the development of designed nucleases that can create site-specific DSB in plant genomes (Christian et al., 2010; Jinek et al., 2012; Li et al., 2011; Shukla et al., 2009; Townsend et al., 2009). Further, as DSB are often repaired by nonhomologous end joining (NHEJ) mechanisms (Gorbunova and Levy, 1997), these DSB reagents have become robust tools of targeted mutagenesis. While the DNA sequence of the DSB site, repaired through NHEJ, cannot be predicted, occurrence of a high rate of short insertion–deletion allows efficient production of variant alleles, which could serve as a rich source of genetic variation. For a more predictable change in the gene sequence, a unique approach of gene editing based on the application of oligonucleotides had been described (Cole-Strauss et al., 1996), which also received a ‘helping hand’ from designed nucleases in enhancing the editing efficiency through DSB induction. Thus, the development of designed nucleases has unleashed an era of crop improvement through genome editing, most notably, trait development by targeted mutagenesis, which is a more precise way of generating useful alleles than the traditional random mutagenesis approaches that introduce numerous genomewide mutations. Designed nucleases are also promising for targeted transgene integration or gene replacement. However, improving HR rates and recovering ‘true’ HR clones are some of the issues still facing the scientific community. Site-specific recombinases derived from yeast or phages have solved the bottleneck of serial targeting, albeit into pre-integrated recombination sites. These developments could enable gene stacking in plant chromosomes, which in turn could facilitate rapid deployment of new traits into diverse crop varieties. The last decade has seen many exciting developments in genome editing capabilities that have significantly impacted both basic and applied aspects of plant biotechnology research and are poised to unleash an era of crop improvement through targeted mutagenesis, precise gene editing, multigene transformation and gene stacking. In this special issue, leading experts in the field of plant genome editing summarize some of the major developments associated with this rapidly moving area. Holger Puchta (2015), one of the pioneers in plant genome editing, provides a personal account of some of the early observations that led to the current paradigm of taking advantage of the cell's natural ability to repair DNA via the creation of targeted double strand breaks. Ow (2015), Lee et al. (2015) and Rivera-Torres and Kmeic (2015) provide comprehensive reviews of the historical aspects of recombinase-mediated transformation, designed nucleases and single-stranded DNA oligonucleotides, respectively, for targeted genome modification. Taken together, these reviews give the reader a detailed perspective of the evolution of this exciting field. Looking towards the future, Srivastava and Thomson (2015) examine the use of recombinases for transgene stacking, Weeks et al. (2015) review the various types of designed nucleases and their use in genome editing across a broad spectrum of plant species, and Sauer et al. (2015) describe targeted mutagenesis using oligonucleotides. Petolino and Kumar (2015) outline how designed nucleases can address some of the issues associated with the deployment of transgenes for commercial trait development, and Wolt et al. (2015) discuss some of the regulatory aspects of genome-edited crops. These reviews paint an optimistic picture of the future of genome editing tempered by the realization that additional knowledge and continued technical developments are required to fully meet its early promise. Finally, Zhu et al. (2015) describe some recent results using the CRISPR/Cas9 nuclease system to mutate genes in Arabidopsis. They found that expressing the nuclease in germ-line cells using a male gametophyte-specific promoter, SPOROCYTLESS, resulted in a higher frequency of diverse, nonchimeric, heritable mutations as compared to constitutive expression. There is little doubt that the field of genome editing will continue to expand and recombinases, designed nucleases and oligonucleotides will play key roles in studies of functional genomics as well as applied crop improvement. The editors feel that this collection of articles will catalyse the continued advancement of this field.