Trait stacking via targeted genome editing

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high‐efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular ‘trait landing pads’ (TLPs) that allow ‘mix‐and‐match’, on‐demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease‐driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS?‐mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo‐derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease‐driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN‐mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.     

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T₁ progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.     

植物转基因整合座位的结构

通过农杆菌和直接DNA转移技术所获得的转基因植株都具有复杂的转基因座位, 转基因整合染色体和染色体区段是随机的, 但组织培养的选择作用表现为非随机性, 偏向整合于染色体的基因富集区。转基因座位除少数含有完整的单拷贝转基因之外, 大多数转基因座位中外源转基因片段、基因组片段和填充DNA相间而存在。转基因座位中转基因及基因组DNA片段产生缺失、重复和染色体的重排, 转基因的完整性对转基因表达具有重要作用。     

Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion

Targeted Genome Optimization (TGO) using site‐specific nucleases to introduce a DNA double‐strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)‐mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre‐existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.     

Integration and inheritance of transgenes in crop plants and trees

Transgene integration and inheritance have been investigated in a number of crop plants and few tree species. Transgene integration is predominantly a random process, whether mediated by Agrobacterium or particle bombardment. Depending on the genomic position of the integrated transgene and structure of the integration site as well as copy number of the transgene in the genome, its expression may be stable or variable. Therefore, integration patterns would affect the mode of transgene inheritance in plants, regardless of the method of gene transfer. So far, both Mendelian and non-Mendelian inheritance of transgenes has been reported across several generations (T1–T3) of crop plants. In few tree species (apple, poplar, plum, and American chestnut), mostly Mendelian inheritance of the transgenes has been observed in the T1 or BC1 generations. However, detailed studies in the transgenic papaya trees showed Mendelian segregation of the transgene in the T1 generation but non-Mendelian inheritance in the T2 generation. Variation in transgene inheritance was also detected in transgenic apple and plum trees. Long generation cycles in many economically important tree species preclude investigation of inheritance of transgenes in the tree progeny. Production of early flowering trees, either by genetic modification or by environmental modulation, would facilitate the study of transgene inheritance across generations of transgenic trees. In order to overcome problems of randomness of transgene integration, targeted transgene insertions by homologous or site-specific recombination or by designer recombinases or nucleases offer prospects for stable integration of transgenes in predetermined locations in the plant genome. And perhaps, that might provide a platform for stable expression and Mendelian inheritance of transgenes in plants.     

Targeted molecular trait stacking in cotton through targeted double‐strand break induction

Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor‐made specificities to introduce a DNA double‐strand break (DSB) at specific target loci. A re‐engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton. The combination of targeted DNA cleavage and homologous recombination–mediated repair made precise targeted insertion of additional trait genes (hppd, epsps) feasible in cotton. Targeted insertion events were recovered at a frequency of about 2% of the independently transformed embryogenic callus lines. We further demonstrated that all trait genes were inherited as a single genetic unit, which will simplify future multiple‐trait introgression.     

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.     

Transgene inheritance in plants genetically engineered by microprojectile bombardment

Microprojectile bombardment to deliver DNA into plant cells represents a major breakthrough in the development of plant transformation technologies and accordingly has resulted in transformation of numerous species considered recalcitrant toAgrobacterium- or protoplast-mediated transformation methods. This article attempts to review the current understanding of the molecular and genetic behavior of transgenes introduced by microprojectile bombardment. The characteristic features of the transgene integration pattern resulting from DNA delivery via microprojectile bombardment include integration of the full length transgene as well as rearranged copies of the introduced DNA. Copy number of both the transgene and rearranged fragments is often highly variable. Most frequently the multiple transgene copies and rearranged fragments are inherited as a single locus. However, a variable proportion of transgenic events produced by microprojectile bombardment exhibit Mendelian ratios for monogenic and digenic segregation vs events exhibiting segregation distortion. The potential mechanisms underlying these observations are discussed.     

Targeted transgene integration in plant cells using designed zinc finger nucleases

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.     

Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.     

Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus

Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.     

The distribution of transgene insertion sites in barley determined by physical and genetic mapping

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.     

Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs

Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection‐mediated gene transfer (ICSI‐MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI‐MGT yielded significantly more embryos with non‐mosaic transgene expression (P < 0.01). In the ICSI‐MGT group, 61.5% (24/39) of the embryos were EGFP‐positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP‐positive in the PN‐injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI‐MGT was transgenic, suggesting that ICSI‐MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI‐MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple‐copy, single‐site integration of a transgene is the primary outcome of ICSI‐MGT in the pig and that ICSI‐MGT is less likely than PN injection to cause transgene integration in a mosaic manner. Mol. Reprod. Dev. 79: 218–228, 2012. © 2011 Wiley Periodicals, Inc.     

Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking.

Transgenes can affect transgenic mice via transgene expression or via the so-called positional effect. DNA sequences can be localized in chromosomes using recently established mouse genomic databases. In this study, we describe a chromosomal mapping method that uses the genomic walking technique to analyze genomic sequences that flank transgenes, in combination with mouse genome database searches. Genomic DNA was collected from two transgenic mouse lines harboring pCAGGS-based transgenes, and adaptor-ligated, enzyme restricted genomic libraries for each mouse line were constructed. Flanking sequences were determined by sequencing amplicons obtained by PCR amplification of genomic libraries with transgene-specific and adaptor primers. The insertion positions of the transgenes were located by BLAST searches of the Ensembl genome database using the flanking sequences of the transgenes, and the transgenes of the two transgenic mouse lines were mapped onto chromosomes 11 and 3. In addition, flanking sequence information was used to construct flanking primers for a zygosity check. The zygosity (homozygous transgenic, hemizygous transgenic and non-transgenic) of animals could be identified by differential band formation in PCR analyses with the flanking primers. These methods should prove useful for genetic quality control of transgenic animals, even though the mode of transgene integration and the specificity of flanking sequences needs to be taken into account.     

2004 SIVB Congress Symposium Proceeding: Transgene Management via Multiple Site-specific Recombination Systems

Summary Current methods for creating transgenic varieties are labor and time intensive, comprised of the generation of hundreds of plants with random DNA insertions, screening for the few individuals with appropriate transgene expression and simple integration structure, and followed by a lengthy breeding process to introgress the engineered trait into cultivated varieties. Various modifications of existing methods have been proposed to speed up the different steps involved in plant transformation, as well as a few add-on technologies that seek to address issues related to biosafety or intellectual property. The problem with an assortment of independently developed improvements is that they do not integrate seamlessly into a single transformation system. This paper presents an integrated strategy for plant transformation, where the introduced DNA will be inserted precisely into the genome, the transgenic locus will be introgressed rapidly into field varieties, the extraneous transgenic DNA will be removed, the transgenic plants will be molecularly tagged, and the transgenic locus may be excised from pollen and/or seed.     

Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR

The generation of transgenic lines is vital to many genetic strategies and provides useful reagents for cell labeling and lineage-tracing experiments. Transposon-based systems offer simple, yet robust, platforms for transgenesis in the frog. Here, we provide a protocol for a microinjection-based transposon transgenesis method using a 'natural breeding' strategy for the collection of Xenopus tropicalis embryos. This method uses co-injection of a plasmid containing a transposon substrate together with synthetic mRNA encoding the transposase to achieve efficient integration of the transgene in the frog genome. We also describe a modified extension primer tag selection linker-mediated PCR technique to identify transposon integration sites within the host genome. This cloning strategy allows rapid identification of genomic sequences flanking the integration sites and multiple independently segregating transposon integration events in a single tadpole can be cloned simultaneously.     

Progress of targeted genome modification approaches in higher plants

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.