Vibha Srivastava

Biolistic mediated site-specific integration in rice

Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to a second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.

Single-copy primary transformants of maize obtained through the co-introduction of a recombinase-expressing construct.

We describe a variation of the method to generate single-copy transgenic plants by recombinase-mediated resolution of multiple insertions. In this study, a transgene construct flanked by oppositely oriented lox sites was co-bombarded into maize cells along with a cre-expressing construct. From analysis of the regenerated plants, a high percentage of the primary transformants harbored a single copy of the introduced transgene, and among these, a majority also lacked the cre construct. We deduce that the expression of cre must have contributed to resolving concatemeric molecules either prior to or after DNA integration into the maize genome.

Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile “transgene maintenance and control” strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.

Physiological and Molecular Basis of Acetolactate Synthase- Inhibiting Herbicide Resistance in Barnyardgrass (Echinochloa crus- galli)

Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target-site or non-target-site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed Ala₁₂₂ to Val and Ala₁₂₂ to Thr substitutions in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of ¹⁴C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. ¹⁴C-Penoxsulam translocation out of the treated leaf (≤2%) was similar among all biotypes. ¹⁴C-Bispyribac-treated AR1 and MS1 translocated 31- 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. ¹⁴C-Imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1.

Site‐specific gene integration in rice genome mediated by the FLP–FRT recombination system

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.

Improved FLP Recombinase, FLPe, Efficiently Removes Marker Gene from Transgene Locus Developed by Cre–lox Mediated Site-Specific Gene Integration in Rice

Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP–FRT system for excising marker gene from the transgene locus developed by Cre–lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.

Marker‐free site‐specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.

Site-specific gene integration technologies for crop improvement

Targeted integration of foreign genes into plant genomes is a much sought-after technology for engineering precise integration structures. Homologous recombination-mediated targeted integration into native genomic sites remained somewhat elusive until made possible by zinc finger nuclease-mediated double-stranded breaks. In the meantime, an alternative approach based on the use of site-specific recombination systems has been developed which enables integration into previously engineered genomic sites (site-specific integration). Follow-up studies have validated the efficacy of the site-specific integration technology in generating transgenic events with a predictable range and stability of expression through successive generations, which are critical features of reliable and practically useful transgenic lines. Any DNA delivery methods can be used for site-specific integration; however, best efficiency is mostly obtained with direct DNA delivery methods such as particle bombardment. Although site-specific integration approach provides unique advantages for producing transgenic plants, it is still not a commonly used method. The present article discusses barriers and solutions for making it readily available to both academic research and applicative use.

Editing Plant Genomes: a new era of crop improvement.

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 sitespecific 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. Sitespecific 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.

Gene stacking by recombinases

Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site-specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high-efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking.