Transposase-assisted target-site integration for efficient plant genome engineering.

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Site-directed integration of exogenous DNA into the soybean genome by LbCas12a fused to a plant viral HUH endonuclease.

High efficiency site-directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR-associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR-associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70-nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site-directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four-fold higher than in untethered controls. Integrations via canonical homology-directed repair or non-homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN-associated tethering to improve site-directed DNA integration in plants.

Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

KEY MESSAGE: Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens.

Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.

A novel real-time polymerase chain reaction method for high throughput quantification of small regulatory RNAs.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are important players of both transcriptional and post-transcriptional gene silencing networks. In order to investigate the functions of these small regulatory RNAs, a system with high sensitivity and specificity is desperately needed to quantitatively detect their expression levels in cells and tissues. However, their short length of 19-24 nucleotides and strong similarity between related species render most conventional expression analysis methods ineffective. Here we describe a novel primer for small RNA-specific reverse transcription and a new TaqMan technology-based real-time method for quantification of small RNAs. This method is capable of quantifying miRNA and siRNA in the femtomolar range, which is equivalent to ten copies per cell or fewer. The assay has a high dynamic range and provides linear readout of miRNA concentrations that span seven orders of magnitude and allows us to discriminate small RNAs that differ by as little as one nucleotide. Using the new method, we investigated the expression pattern of gma-miRMON1, a novel miRNA identified from soybean leaves. The results were consistent with our results obtained from Northern blot analysis of gma-miRMON1 and Affymetrix microarray analysis of the gma-miRMON1 precursor, suggesting that the new method can be used in transcription profiling.

Modifying lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing transgene cassette.

Although it is one of the major crops in the world, corn has poor nutritional quality for human and animal consumption due to its low lysine content. Here, we report a method of simultaneous expression of a deregulated lysine biosynthetic enzyme, CordapA, and reduction of a bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharophine dehydrogenase (LKR/SDH), in transgenic corn plants by a single transgene cassette. This is accomplished by inserting an inverted-repeat sequence targeting the maize LKR/SDH gene into an intron of a transgene cassette that expresses CordapA. This combination of LKR/SDH silencing and CordapA expression led to the accumulation of free lysine to over 4000 p.p.m. in transgenic corn grain, compared to less than 100 p.p.m. in wild-type controls. This intron-embedded silencing cassette design reduces the number of transgene cassettes needed in transgenic approaches for manipulating metabolic pathways that sometimes require expression of one gene and silencing of another.

RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants.

A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation.

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.

A test for ectopic exchange catalyzed by Cre recombinase in maize.

A maize line expressing Cre recombinase as well as the recipient line without the transgene were assayed for evidence of ectopic recombination within the maize genome. Such a test is valuable for understanding the action of Cre as well as for its use to recombine two target lox sites present in the chromosomes. Pollen examination and seed set tests of material expressing Cre provided no evidence of ectopic recombination, which would be manifested in the production of translocations or inversions and result in pollen abortion and reduced seed set. Root-tip chromosome karyotype analysis was also performed on material with and without Cre expression. Chromosomal aberrations in Cre+ material were not observed above the background level.

Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors.

By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome. This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation. In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders. These results also provide evidence that both the right and left borders can initiate and terminate T-strands. Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions.