Progress 09/01/20 to 08/31/24
Outputs
Target Audience:Our target audience for this reporting period was professors, researchers, postdocs, industry members, students, government regulatory bodies, and the general public. These were reached through internal scientific meetings at OSU and with collaborating research and industry groups, as well as lectures and invited talks. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the final reporting period, the project has enabled PD Goralogia to give an invited talk about plant transformation improvement to a multiagency panel sponsored by DOE in 2023, and to attend the annual Society for in vitro Biology (SIVB) and IUFRO Tree Biotechnology meeting. It has also provided the opportunity for PD Goralogia to collaboratively interact and share tools with USDA-ARS groups (tools built in this project are being trialed in six academic, government, and private research groups). One new student from the OSU URSA/Engage program worked on the project in 2024, and gave multiple internal presentations at campus events. Over the course of the project, five presentations at national conferences were enabled through grant funds, multiple publications, and a filed patent were accomplished which contributed to training and professional development for Goralogia and trainees associated with the grant. How have the results been disseminated to communities of interest?During the finalreporting period, we have made efforts to reach our target audiences through 5 oral presentations, discussions with industry and USDA collaborators, and education activities via OSU classes. A description of progress was provided to industrial collaborators of our GREAT TREES research consortium on August 4th of 2024. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim I- In initial stages of the project, we investigated various Cre induction systems in poplar for mediating transgene deletion during early regeneration. This included chemical induction (dexamethasone and beta-estradiol), developmental induction (cell-specific promoters) and environmental induction (optogenetic light and heat-shock). To detect positive signals of an excision event, rather than relying on loss of a fluorescent reporter signal, we developed a reporter which switched from RFP to GFP expression. In total, the induction system had a small effect on excision outcomes, and shoot excision rates were low (0-2% per construct). The top shoot excision performers were a combination of developmental-chemical induction (pCSP3:Cre-GR, expressed in early shoot primordia), and heat shock induction (pGm17.5:Cre, derived from soybean). Extensive silencing where neither the RFP nor switched GFP were expressed was frequently observed in resultant shoots. These results led us to focus on fundamental aspects of Cre recombination in plants, rather than expression control alone. Results from other groups suggested DNA methylation was occurring in somatic cells after Cre expression, preventing efficient excision and causing transgene silencing. This was confirmed after 5-azacytidine treatment of Cre expressing transgenic poplars, which recovered expression of the fluorescent markers after treatment. Treatment with several concentrations of 5-azacytidine did not improve excision in Cre transgenics, unfortunately. Another report [PMID: 33742463] highlighted the inhibitory effects of DNA methylation on bases present within the lox recombination spacer sequence in vitro, and we decided to follow up on this research by developing a screening system to find sites with high affinity for Cre recombinase which lacked any C/G bases in the spacer. We screened and sequenced these sites in E. coli using a degenerate primer strategy and tested several sites in a tobacco agroinfiltration system. An efficient cytosine-free spacer, loxW7 was found. Tests using agroinfiltration in N. benthamiana showed strongly elevated rates of successful excision using loxW7 vs. conventional loxP. To further investigate plant-optimized lox sites for improved excision in somatic cells, we developed a second reporter system which improved on the RFP-GFP switch construct. This strategy used a red fluorescent protein reporter whose coding sequence is separated by two introns, with an intervening exon encoding an inflexible linker which is removed by excision. Using this reporter, we transformed poplars with constructs containing the reporter but without Cre recombinase, with recombinase and conventional loxP sites, or with Cre recombinase but containing cytosine-free lox sites (loxW7). Under a constitutive promoter we found a 53% reduction in transgenic shoot recovery in loxP containing constructs vs. controls, with 57% of product loxP shoots being silenced for both reporters. LoxW7 containing constructs had only a 7% reduction in transgenic shoot recovery. The loxW7 sites were still capable of producing successfully recombined, fluorescent events at a rate of 15% of recovered shoots, which suggests strongly reduced silencing outcomes using these sites. Using the heat-shock promoter driven Cre, we saw similar effects, with 52% reduction in shoot production in loxP constructs, and only 20% in loxW7 constructs vs. controls. Additionally 12% of loxW7 events were excised before heat shock induction ("overswitched"), vs. 51% after heat shock. For loxP, 6% of shoots were excised prior to heat shock. Overall, these experiments suggest that loxP containing-reporters have strong Cre-triggered effects which reduce transgenic event recovery and inhibit recombination outcomes, likely through DNA methylation activity. New lox sites, in addition to the wide variety of Cre control systems, offer expanded capabilities for transgene excision systems in plants. Aim II- In initial stages of the project, we developed a suite of binary vectors to enable gene editing through CRISPR-Cas9 and subsequent excision using loxP sites flanking the transgene. The first phases of the project used a developmental-chemical induction scheme for Cre recombinase (pCSP3:Cre-GR). Testing with PDS showed that while chlorotic product shoots indicative of mutations could be obtained at high rates, they were difficult to maintain in vitro for subsequent analysis and thus we decided to focus on LFY and GAI as target genes. A large set of transformations including more than 200 events with two editing constructs was completed. We first screened the population for successful editing at the target loci but were only able to identify three events with editing and complete transgene excision. A population of approximately twenty edited events was isolated for further study, which lacked GFP expression but were confirmed by PCR to still be transgenic. Repeated treatments with dexamethasone and 5-azacytidine were not effective at inducing subsequent transgene excision. After progress had been made to identify novel plant-optimized lox sites to employ in our editing constructs, we sought to test these in combination with gene editing in poplar. To test the new cytosine-free lox sites (loxW7) combined with heat shock Cre induction for its utility in gene editing, we transformed two poplar genotypes with a complex construct using these approaches that combines morphogenic genes, gene editing, and transgene excision. One component of this construct was rol genes isolated from Agrobacterium rhiozogenes, whose purpose was to initiate hairy root clones at rapid and efficient rates in woody plants. A second component was genes to promote inducible shoot formation from hairy root clones. To do this we included the WUSCHEL and ipt genes, expressed under heat shock promoters. We also included Cas9, gRNAs to target the LFY and GAI genes, reporter genes, and our excision machinery. We achieved high rates of hairy root formation using this approach, with transgenic hairy roots obtained in 40-55% of explants in each genotype. Shoot formation after heat shock induction and placement on shoot-induction media was high using a 4hr/day 38C heat shock treatment with a two-week duration, with 68% of hairy root explants producing at least one shoot. We obtained fully excised events in 32% or 37% of shoots tested in each genotype, respectively. Among the events 100% showed evidence of editing with ~50% edited allele frequency in at least one of the three gRNA sites, particularly at the GAI target gene. Overall, our work using this construct shows that usable rates of transformation, gene editing, and excision can be accomplished in somatic tissues of a woody, clonally propagated plant using Cre recombinase using our approach. Aim III- Primarily during years two and three of the project, we worked to assess any Cre toxicity in transgenic poplars. To look for recombinase-specific toxic effects we performed a greenhouse study using ten independent Cre-overexpression poplar events with at least ten clonal ramets of each event. We measured height and diameter of the trees after three months of growth, in addition to other traits (SPAD, leaf area, branching etc.) and found no significant differences to wild-type and empty vector controls, and no morphological differences. To characterize the fully-excised, edited event footprints and any effects they had on the genes surrounding the inserted footprint, we selected five events which met our criteria from the hairy root excision construct experiment. Despite many attempts we were unable identify the footprint location in the genome using TAIL-PCR. Due to these technical limitations, we have decided to prioritize events for long-read whole genome sequencing (Oxford Nanopore) to identify the inserted footprint site(s), and further characterize local gene expression, which is currently ongoing.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Goralogia GS, Andreatta IM, Conrad V, Xiong Q, Vining KJ, Strauss SH. Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9. Front Bioeng Biotechnol. 2024 Jun 21;12:1412927. doi: 10.3389/fbioe.2024.1412927. PMID: 38974658; PMCID: PMC11224489.
- Type:
Other Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Goralogia GS, Willig C, Strauss SH. Engineering Agrobacterium for improved plant transformation. The Plant Journal. 2024
- Type:
Other Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Goralogia GS, Ma C, Taylor DS, Lawrence A; Conrad V, Peremyslova E, Strauss SH. Co-transformation using T-DNA genes from Agrobacterium strain 82.139 enhances regeneration of transgenic shoots in Populus. Plant Biotechnology Journal. 2024
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Agrobacterium T-DNA genes as tools to promote regeneration of transgenic woody plants. SIVB 2024 World Congress on in vitro Biology, St. Loius, MO, June 2024
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Back to the Future: A co-transformation approach with Agrobacterium T-DNA genes promotes regeneration of transgenic woody plants. IUFRO Tree Biotechnology, Annapolis, Maryland, August 2024
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Excisable gene editing systems: Generation of dwarf and sterile poplars using a developmental and chemical-controlled
CRISPR/recombinase excision system
American Society for Plant Biology Annual Meeting, 2021, Lightning talk
Goralogia, G.S. and Steven H. Strauss. https://biotechlab.forestry.oregonstate.edu/sites/default/files/StraussASPB2021.pdf
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Somatic Transgene Excision Strategies for Gene Editing in Clonally Propagated Plants
Society for In Vitro Biology National Meeting, June 2021 (invited, online)
Goralogia, G.S. and Steven H. Strauss.https://biotechlab.forestry.oregonstate.edu/sites/default/files/Strauss%20SIVB%202021%20v7.pdf
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
A developmentally timed transgene excision system for somatic removal of gene editing components in asexually propagated plants
Presented at the American Society of Plant Biologists, Annual Meeting, 27-31 July 2020 (invited, online)
Greg S. Goralogia, et al. https://biotechlab.forestry.oregonstate.edu/sites/default/files/GSG%20ASPB%202020%20FINAL-compressed.pdf
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Low rates of non-target mutations in field and greenhouse-grown CRISPR/Cas9 expressing transgenic trees
Poster presented at American Society for Plant Biology Annual Meeting, Portland, Oregon, 2022
Greg S. Goralogia, Isabella M. Andreatta, Qin Xiong, Kelly J. Vining, Estefania Elorriaga, Cathleen Ma and Steven H. Strauss
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Off-target mutations in CRISPR/Cas9-expressing transgenic trees engineered for containment
International Society for Biosafety Research Congress May 2023, St. Louis, MO, USA
Greg Goralogia and Steven H. Strauss
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Goralogia, G.S., Redick, T.P. & Strauss, S.H. Gene editing in tree and clonal crops: progress and challenges. In Vitro Cell.Dev.Biol.-Plant 57, 683699 (2021). https://doi.org/10.1007/s11627-021-10197-x
|
Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Our target audience for this reporting period was professors, researchers, postdocs, industry members, students, government regulatory bodies, and the general public. These were reached through internal scientific meetings at OSU and with collaborating research and industry groups, as well as lectures and invited talks. Changes/Problems: Aim1b included planned experiments which would edit a GFP or DsRed gene in previously created poplar transgenics, then induce excision of the editing transgene. Originally it was hoped this approach would enable easy detection of editing and excision together through visual markers. One unexpected issue we discovered is that having multiple reporters in the editing transgene is very useful as these constructs are large and can be subject to silencing during the Cre-mediated excision process. Having both DsRed and GFP at either end of the T-DNA helps to identify partially integrated events and have a second failsafe marker if an event becomes silenced during excision (this prevents mis-interpretation of editing/excision results). Because our sequencing protocols to identify edits at the LFY and GAI target loci in poplars are nowstreamlined, we have opted to focus our efforts on editing/excision analysis on the native poplar targets. We do not anticipate this change will affect an accurate assessment of the excision and editing rates inherent in the system. What opportunities for training and professional development has the project provided?During this reporting period, the project has enabled PD Goralogia to give an invited guest lecture, and to attend the American Society of Plant Biologists (ASPB) Western Regional Meeting. It has also provided the opportunity for PD Goralogia to collaboratively interact and share tools with USDA-ARS groups. It has also provided the opportunity for Co-PI Strauss to discuss "clean" gene editing technology for forest trees within the context of biosafety and new transformation technologies (ISBR 2023, and PlantGENE annual meeting). One new student from the OSU URSA/Engage program worked on the project in 2023 and will continue to be involved under no-cost extension in 2024 (https://academicaffairs.oregonstate.edu/research/ursa-engage). How have the results been disseminated to communities of interest?During this reporting period, we have made efforts to reach our target audiences through 3 oral presentations (including one to a plant-transformation scientific network), discussions with industry and USDA collaborators, and education activities via OSU classes. A description of progress was provided to industrial collaborators of our GREAT TREES research consortium on December 7th of 2022 and September 12th, 2023. What do you plan to do during the next reporting period to accomplish the goals?Aim I: In the next reporting period, we will complete the analysis of stable transgenic recovery and excision rates using loxP and a synthetic, cytosine-free lox site (loxW7) coupled with heat shock inducible or constitutively expressed Cre recombinase using both of our fluorescence detection systems (tdTomato>>GFP, mScarlet3off>>on), and complete ddPCR T-DNA copy number analysis. We expect these experiments to be finished by February 2024. Aim II: In the next reporting period under no-cost extension, we will complete the large-scale editing/excision study to generate semi-dwarf/sterile poplars using heat-shock inducible Cre and a synthetic cytosine-free lox site. Aim III: By Spring 2024 we will find the insertion footprint sites in our paragon edited and excised events for downstream analysis, whether by TAIL-PCR or by targeted long-read sequencing (nanopore). By Summer 2024 we will complete gene expression analysis and chromatin effects on genes surrounding the excision footprint insertion site.
Impacts
What was accomplished under these goals?
Aim I (90% complete): In previous years, we used a fluorescent switch reporter system (tdTomato>>GFP after recombination) to assess excision outcomes in stable transgenic poplars under different induction schemes. Experiments with various light, chemical and heat induction systems to induce Cre recombinase in prior work showed the soybean HSP17.5 heat-shock promoter to be the most effective in poplar transformation and regeneration. To further improve our excision detection transgene architecture and confirm our excision strategies were robust to changes in vector design, we also developed a parallel detection system. This system uses an RFP gene (mScarlet3) whose coding sequence is separated by two introns. Within the introns are embedded lox sites, which concatenate together upon excision. Between the internal introns is a synthetic exon which encodes an inflexible linker, separating the two halves of the fluorescent protein. Thus prior to excision, an improperly folded mScarlet3 protein is produced, and after excision, the protein is correct and red fluorescence is observed. This system yields approximately three times greater brightness after excision and has undetectable background fluorescence in the off state (without Cre). This architecture provides an alternative to the gene deletion shift (tdTomato>>GFP after recombination) where Cre binding to lox sites near the transcription start site may interfere with GFP or tdTomato expression in some events. To test excision rates in stable transgenic plants using both of these reporters we transformed poplars with constitutive and heat-shock inducible Cre genes. We also compared the same constructs with traditional loxP recognition sites and a synthetic lox site lacking cytosines in the core recombination region, which we hypothesized to be more efficient due to reduction of DNA-methylation activity by Cre (see below). Though some of these experiments are currently ongoing, an analysis of the constitutively expressed Cre system showed that novel engineered lox sites could improve excision rates from 1% or less to10% of recovered shoots. Because previous reports determined single copy T-DNA insertions have an increased likelihood of successful excision, excised and unexcised shoots are being further analyzed using digital droplet PCR (ddPCR) to determine T-DNA copy number. This will help us to better understand whether unexcised events are predominantly multi-copy events. We have made significant efforts reduce transgene silencing after Cre activation (discussed in prior reporting period Changes/Problems; see PMID: 33742463 for related study). Two tools of focus to manipulate the silencing/DNA methylation response included the viral P19 silencing suppressor and the viral βC1 (PMID:35102164) REPRESSOR OF SILENCING 1 (ROS1) interacting protein. In tobacco transient assays, P19 and βC1 co-expression greatly increased Cre-mediated excision of the tdTomato>>GFP reporter, however in stable transgenic poplars they together strongly reduced shoot regeneration rates when expressed under the strong pAtUBQ10 promoter. Future work with these tools will likely focus on inducible expression systems. As discussed in the prior reporting period, we also developed a screening method to produce synthetic lox sites which might be less susceptible to DNA methylation of cytosines in the core crossover region. Of these approximately 30 sites we tested several in vitro to screen for activity with purified Cre recombinase protein. Several sites showed strong activity at about half the rate of loxP, and a top performer "loxW7" was selected for in planta study. In tobacco transient assays it outperformed loxP and became a focus of study in both our fluorescent excision detection system as well as incorporated into our editing and excision vectors for further analysis. Aim II (90% complete): Under this aim we have and will continue to produce edited, excised, semi-dwarf and sterile poplars for use in genomic studies in Aim III. Small scale experiments using multiple Cre induction schemes investigated thus far have produced a suite of excised events which are edited at either the dwarfism (GAI) or sterility (LFY) locus or are excised but unedited for genomic analysis in Aim III. We have selected two edited and excised events for further analysis. Further large-scale transformation studies are in progress which include heat-shock promoter driven Cre and the best performing synthetic lox site (see above). This set of experiments also uses our efficient transformation method which co-delivers hairy root inducing genes in the same T-DNA. To date, we have obtained over 200 independent events in two poplar genotypes with a third genotype of black cottonwood (Populus trichocarpa) ongoing. These are being subjected to heat shock treatments to induce excision and recovered on shoot-inducing media (SIM). We are measuring excision, shoot regeneration, and gene editing rates in the resulting population for a summary of the overall efficiency of our approach for excised and edited poplars. Aim III (70% complete): To assess Cre toxicity we performed a greenhouse study on Cre overexpressing poplars and will perform DNA methylation/gene expression analysis on footprints in the genome of excised and edited lines. To look for recombinase-specific toxic effects we grewten independent Cre-overexpression poplar events with at least ten clonal ramets of each event in the greenhouse alongside similarly-regenerated wild-type controls. We measured height and diameter of the trees after three months of growth, in addition to other traits (SPAD, leaf area, branching etc.) and found no significant differences tocontrols, and no morphological differences. We retained tissue to analyze gene expression of the transgenes and detect if Cre expression is preferentially silenced in the resultant lines due to selection in tissue culture. We are currently working on finding the T-DNA insertion sites in our two selected successfully excised/edited events by TAIL PCR (PMID: 22093809) and are also investigating targeted long-read sequencing approaches to reach the same outcome. After we find the footprint location, we will perform expression analysis by qPCR on genes within 20kb windows of the insertion site, as well as bisulfite-seq relative to controls to determine any lingering effects of the transgene insertion.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Presentation at ASPB Western Section Regional Meeting, April 2023
Unexpectedly divergent off-target mutations found in field- and greenhouse-grown CRISPR/Cas9 expressing transgenic trees
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Presentation at International Society for Biosafety Research Congress, June 2023
Off-target mutations in CRISPR/Cas9-expressing transgenic trees engineered for containment
https://biotechlab.forestry.oregonstate.edu/sites/default/files/Strauss_CRISPR_ISBR2023.pdf
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Presentation at PlantGENE Network Annual Meeting, October 2023
"Altruistic transformation with novel Agrobacterium genes: A model for advancing transformation of recalcitrant plant species?
https://biotechlab.forestry.oregonstate.edu/sites/default/files/StraussGoralogia_October2023.pdf
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Our target audience for this reporting period was professors, researchers, postdocs, industry members, students, government regulatory bodies, and the general public. These were reached through internal scientific meetings at OSU, national society meetings, with collaborating research and industry groups, as well as lectures and invited talks. Changes/Problems: Schedule of Aim Ib was delayed until construct elements ameliorating Cre-mediated silencing were surveyed. These are now in-progress or complete and nearly all have been incorporated into our editing vector systems. For Aims Ib/II, we have mobilized the hairy root inducing rol genes into our GAANTRY editing vectors and have begun using composite plant transformation methods to induce transgenic roots, rather than relying on traditional indirect shoot organogenesis. This method generates transgenic tissues more efficiently (avg. 42% transformation rate vs. approximately 20%) and rapidly (transgenic tissues for propagation within 6 weeks) and enables better visualization of excision during Cre induction with fluorescent reporters. Excision rates are comparable between transformation systems with the same induction scheme. Excised sectors with only the footprint remaining regenerate into phenotypically normal shoots after placement on shoot inducing media. This approach allows us to test the simultaneous use of morphogenic regulator genes during transformation and excision, which is rapidly becoming a common approach, as well as increase our experimental throughput. What opportunities for training and professional development has the project provided?During this reporting period, the project has enabled PD Goralogia to give two invited guest lectures, a short format talk at the USDA-BRAG annual meeting, and attend the American Society of Plant Biologists (ASPB) annual meeting. It has also provided the opportunity for PI Goralogia to collaboratively interact and share tools with one USDA-ARS group. It has also provided the opportunity for Co-PI Strauss to discuss "clean" gene editing technology for forest trees within the context of new transformation technologies (https://people.forestry.oregonstate.edu/steve-strauss/presentations, 2022 IUFRO, SIVB). New students from the OSU URSA/Engage program are expected to work on the project in 2023 (https://academicaffairs.oregonstate.edu/research/ursa-engage). How have the results been disseminated to communities of interest?During this reporting period, we have made efforts to reach our target audiences through one undergraduate thesis publication and defense, 4 oral presentations, discussions with industry and USDA collaborators, and education activities via OSU classes. A description of progress was provided to industrial collaborators of our GREAT TREES research consortium on April 7th and December 7th of 2022. What do you plan to do during the next reporting period to accomplish the goals?Aim Ia: In the next reporting period, we will complete the analysis of the fluorescent excision switch constructs which have Cre controlled by a red-light optogenetic system. We will also complete our survey of T-DNA copy number when delivered by binary vs. vir (GAANTRY) systems by digital droplet PCR. Both of these experiments will be finished by Spring 2023. Aim Ib: In the next reporting period, we will complete the large-scale editing/excision experiment using GFP transgenes as a target, recording the editing and excision rates. This will include vectors with heat-shock Cre induction and any anti-silencing components which aid excision. At present these are flanking Rb7 MARs, P19, and βC1. This will include approximately 200 transgenic events. Aim II: In the next reporting period, we will complete the large-scale editing/excision experiment to generate semi-dwarf/sterile poplars. We will also determine which of the gene silencing prevention tools we developed are most effective for improving excision in stable transgenic poplar. Silencing tools will continue to be analyzed in the fluorescent switch system, and all components with positive excision effects will be included in large editing GAANTRY-launched vectors. Aim III: By September 2023 we will complete long-read sequencing on generated edited/excised events, Cre overexpression events, and controls, and perform targeted DNA methylation and gene expression analyses at excision footprints. By Summer 2023 we will have finished greenhouse data collection on Cre overexpression events. Controls include wild-type poplars and transformation escapes. Traits will include leaf area, dry weight, height, stem diameter and volume, SPAD, and petiole length. If events are regenerated at a fast enough pace and time permits, we will also analyze events which co-express Cre with anti-silencing components in the greenhouse to assess any phenotypic aberrations.
Impacts
What was accomplished under these goals?
Aim Ia (80% complete): We used a fluorescent switch reporter system (tdTomato>>GFP after recombination) to assess excision outcomes in stable transgenic poplars under different induction schemes. Since the prior reporting period we expanded the number of Cre induction systems to include dexamethasone (Dex) hormone induction in a transactivation architecture, blue light-mediated induction (PMID: 33620312) through control of Cre protein, and an optogenetic system using monochromatic red-light activation (PMID: 32601426). Transactivated Dex induction outcomes were slightly less effective than the soybean HSP17.5 heat-shock promoter driven Cre investigated previously. Blue light-mediated induction of Cre had only incomplete excision in developed shoots and had high rates of visible silencing of the tdTomato reporter, suggesting high background activity during early regeneration. Cre induction testing with the red-light optogenetic system, and stable transgenic events are nearly ready for analysis. Previous reports determined single copy T-DNA insertions have an increased likelihood of successful excision. We similarly performed copy number analysis using qPCR on unexcised events after shoot regeneration. These analyses suggest high T-DNA insertion copies (avg=3.1) in our transformation population. These are being further verified using digital droplet PCR (ddPCR), a more reliable method of quantification. Together these data suggest that modern, high copy binary vectors and hypervirulent Agrobacterium strains should be avoided when transgene excision is required. Comparisons of T-DNA copy when delivered from low-copy vir plasmids (GAANTRY system, PMID: 29901840) are presently ongoing. Other contributing factors such as silencing after excision which could skew copy number populations in recovered events are being actively investigated (see Aim II). Aim Ib (30% complete): Here we are editing a fluorescent reporter gene (GFP) which is stably integrated into a target transgenic poplar event and subsequently inducing Cre excision of the editing transgene. Constructs have been developed towards this aim which are: 1) delivered by vir plasmid to reduce copy number, 2) incorporate our most successful heat-shock driven Cre cassette, and 3), incorporate best performing anti-silencing construct elements discussed under Aim II. The transformations will begin in the early phase of year 3 with large scale experiments (producing approximately 200 transgenic events). Aim II (60% complete): Under this aim we have and will continue to produce edited, excised, semi-dwarf and sterile poplars for use in genomic studies in Aim III. Small scale experiments using multiple Cre induction schemes investigated thus far have produced a suite of excised events which are edited at either the dwarfism (GAI) or sterility (LFY) locus or are excised but unedited for genomic analysis in Aim III (Figure 1C,D). Final, large scale transformation studies are about to begin using vector configurations like Aim 1b, which include the heat-shock promoter driven Cre and anti-silencing components. We expect to produce approximately 200 transgenic events for precise assessment of excision and editing rates using the optimized construct architectures. We have made significant investment validating tools which reduce transgene silencing after Cre activation (discussed in prior reporting period Changes/Problems; see PMID: 33742463 for related study). These construct elements include the artificial microRNA-mediated knockdown of the poplar homolog of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a key siRNA pathway component in plants, the co-expression of the viral P19 silencing suppressor, the co-expression of the viral βC1 (PMID:35102164) REPRESSOR OF SILENCING 1 (ROS1) interacting protein, and the use of flanking tobacco Rb7 matrix attachment region (MAR) elements around the delivered T-DNA. In tobacco transient assays, P19 and βC1 co-expression greatly increased Cre-mediated excision of the tdTomato>>GFP reporter (Figure 1A,B). Stable transgenic poplars with similar configurations are currently being generated. The use of flanking Rb7 MAR elements resulted in strong reductions in silencing of fluorescent reporters after Cre induction, but did not significantly change the rates of successful, complete excision among events. Inducible DRM2 knock-down using a heat-shock promoter driven amiRNA resulted in a detectable but statistically insignificant increase in Cre-mediated excision. Constitutive versions of this construct are also being produced and tested. In addition to these tools, we also developed a bacterial screening system to find novel lox recombination sites with reduced capability to be methylated in plants. Constructs containing these modified lox recombination sites are also being tested in stable transgenic poplars alongside other silencing-suppressive components. These tools are being prototyped in our fluorescent switch reporter excision system before being incorporated into GAANTRY-based editing vectors. Aim III (30% complete): To assess Cre toxicity and any DNA damage we will perform a greenhouse study on Cre overexpressing poplars and edited/excised events and perform long-read sequencing (to look at structural variation) and DNA methylation/gene expression analysis on excision footprints in the genome. This reporting period we produced 15 Cre overexpression events needed for our greenhouse study. 10 clonal ramets of each event have been propagated and are hardening for transfer into newly constructed greenhouses at OSU. These events appear phenotypically normal in in vitro culture, as hypothesized. We have obtained access to a local MinION sequencer for performing long-read sequencing and are conducting pilot tests to assess DNA read length by poplar DNA extraction method. Similarly, T-DNA insertion sites in successfully excised events are being analyzed by TAIL PCR (PMID: 22093809) to design bisulfite-seq and gene expression targets and associated amplicons. Listed figures can be viewed at the following link: https://www.dropbox.com/scl/fi/fwpikyudu25kav488u1qs/SITE-SPECIFIC-RECOMBINASES-AS-TOOLS-FOR-PLANT-GENOME-EDITING-EFFICACY-AND-RISK-Figure-2022.pptx?dl=0&rlkey=tc6pl55oa1094y6tynaglrvd8
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Low rates of non-target mutations in fieldand greenhouse-grown CRISPR/Cas9 expressing transgenic trees https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/ASPB2022_Goralogia.pdf
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Of media and miracles: Successes and frustrations in the search for efficient regeneration and transformation methods for trees and crops https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/Nagle_StraussSIVB2022_media_miracles.pdf
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Expression Properties of Two Promoters During Callus and Shoot Development in Transgenic Poplar https://ir.library.oregonstate.edu/concern/honors_college_theses/hx11xp992
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Our target audience for this reporting period was professors, researchers, postdocs, industry members, students, government regulatory bodies, and the general public. Changes/Problems:Describe major changes/problems in approach and reason(s) for these major changes. We made several small construct and transgenic event changes to better suit the project. These included: 1. Improved single T-DNA excision testing vector with a fluorescent switch from tdTomato to GFP. This process created an active signal for excision in individual cells and more closely mimicked the single T-DNA delivery of our editing vectors. 2. Increased the diversity of Cre-excision induction schemes to include constitutive Cre-GR, estradiol induction, and heat shock induction, as initial experiments suggested that dexamethasone induction may not be efficient as expected in early regeneration stages (Aim 1a). 3. We opted to abandon pAtSTM:Cre-GR driven transgenics to compare against CSP3 due to lower expression found in regenerating shoot tissues. 4. We will not be performing PDS editing experiments due to the difficulty and extra time required to obtain sufficient tissue for molecular analysis, and will focus on GFP and target trait editing. 5. Changing the editing target event in Aim 1b to pAtUBQ10:GFP rather than dsRed, as we discovered that loss of fluorescent signal in dsRed expressing plants lasts for several cell divisions presumably due to the increased protein lifetime of dsRed and similar fluorescent proteins. 6. Adding the additional editing target of GAI homologues in addition to LFY to increase the horticultural value of edited/excised products for future regulatory engagement. 7. Investigating the effect of suppression of methylation on regeneration and excision rates, using an amiRNA approach. We hope this may improve Cre efficiency in our constructs. What opportunities for training and professional development has the project provided? Students and PD/Co-PI's working on this project have attended conferences (SIVB/ASPB), given presentations, disseminating their research and providing them with valuable networking opportunities. This also included two students who partook in the local Oregon State University URSA/ENGAGEprogram (academicaffairs.oregonstate.edu/research/programs/ursa-engage), with two associated presentations by students Anna Brousseau(www.dropbox.com/s/k382qaglztz53qk/Anna%20Brousseau%20URSA%20Meeting%20Presentation%202021.pptx?dl=0)and Daniel Casey-Hain(www.dropbox.com/s/785j4nqgfyb67hu/Daniel%20URSA%202021%203%20min%20revised.pptx?dl=0), and training for a third student in molecular analytical techniques. This grant provided opportunity to publish a review article published in In Vitro Cell and Developmental Biology(cited). How have the results been disseminated to communities of interest? During this reporting period, we have made efforts to reach our target audiences through one review publication, 5 posters and oral presentations, discussions with industry collaborators, and education activities via OSU classes. A description of progress was provided to industrial collaborators of our GREAT TREES(people.forestry.oregonstate.edu/steve-strauss/great-trees-cooperative)research consortium on February 5 and August 19 of 2021. What do you plan to do during the next reporting period to accomplish the goals? Aim 1: Develop an efficient transgene excision and gene editing system Sub-aim 1: Optimization of DEX induction of Cre under different promoters and developmental stages. By the next reporting period, we will investigate Cre induction and excision efficiency using our stable transgenic tdTomato/GFP switch reporter transgenic hybrid poplars. Using different induction conditions on mock and treated samples. We expect these to be complete by the next reporting period. We will also finish making GAANTRY (not binary delivered) similar reporter constructs for pAtCSP3:Cre-GR to compare against binary delivered transgenics already produced, and classify any difference in T-DNA copy number measured by qPCR between delivery strategies. We will pursue similar excision treatments to compare against binary systems. Sub-aim 1b: Testing of editing in combination with excision to determine overall system efficiency Using the best chemical induction approaches developed in the next reporting period under Aim 1a, we will transform and classify editing / excision outcomes on constructs transformed in the pAtUBQ10:GFP transgenics. Given our experience over the first reporting period, we intend these constructs to be GAANTRY delivered constitutive dexamethasone inducible constructs. We will produce a minimum of 20 transgenic events using this approach. Aim 2: Production and genomic assessment of edited/excised poplars We expect to obtain the constitutive Cre (pAtUBQ10:Cre), control, escape, and edited/excised events required for long read sequencing and footprint analysis in Aim 3 by the end of the next reporting period. Once the excision analysis on the second GAI/LFY transformation is complete we will prepare the results for publication in the upcoming reporting period. Aim 3: Test if excision footprint affects local gene expression and chromatin We expect to obtain enough materials to begin preparations for DNA extraction for long-read sequencing and begin TAIL-PCR analysis to find footprint genomic locations in key events by the end of the next reporting period. This will allow us to be on track for footprint analysis and genomic integrity analysis in year three of the grant.
Impacts
What was accomplished under these goals?
Aim 1: Develop an efficient transgene excision and gene editing system Subaim 1a: Optimization of DEX induction of Cre under different promoters and developmental stages. (50% completion) Listed figures are available at this link: https://www.dropbox.com/s/tvs57xmuzwqklqk/BRAG%20OSU%202021%20Progess%20Report%20Figures%20v2.pdf?dl=0 To test the optimum DEX induction and promoter systems for Cre recombinase expression, we assembled constructs and transformed 717 hybrid poplars. We made several small construct revisions from our initial proposal to more closely mimic the gene editing construct used later in Aims 1 and 2. This included delivering constructs as a single T-DNA to better understand transgene silencing during Cre induction and an improved "active excision" fluorescent protein system where cells express tdTomato until excision when they begin to express GFP and lose tdTomato by Cre-mediated deletion (Figure 1A,B). Due to the changes in construct architecture and assembly methods, as well as fluorescence detection in our laboratory, we were able to expand the numbers of excision methods investigated. These include constitutive dexamethasone induction, developmental dexamethasone induction, constitutive transactivated estradiol induction, and heat shock induction. We were able to generate a minimum of 20 events for all construct types for future experimentation of excision rates. To test excision during initial transformation we performed tests on populations of 50-70 explants per treatment, which contained transgenic callus produced de novo during our transformation procedure. While excision rates of some cells during early callus and shoot formation were high (42% for 35S:Cre-GR and 58% for CSP3:Cre-GR), complete structure (callus/shoot) transformation rates were low in treated populations compared to controls when dexamethasone was supplied in culture media on a explant level (30% for 35S:Cre-GR and 2% for CSP3:Cre-GR) (Figure 1C). Heat shock promoter driven Cre events had similar levels of conversion compared to dexamethasone induction constructs in early regeneration. Transgene silencing was evident in heat shock and constitutive dexamethasone construct types, where fluorescent signal was lost in regenerated calli. We are in the process of obtaining enough in vitro material to begin dexamethasone induction testing for best method and concentration on explants in the early stages of root and shoot regeneration. We have begun characterizing the copy number of Cre excision reporter constructs to compare excision outcomes against this metric, as we hypothesize integration complexity negatively impacts Cre excision outcomes on an event-by-event basis. To complement this, we also developed similar Cre excision reporter constructs mobilized in the GAANTRY vector system, which launches T-DNAs directly from the Agrobacterium vir plasmid. We anticipate these constructs will be useful comparators to binary delivered T-DNAs by reducing the insertion copy number and integration complexity. Subaim 1b: Testing of editing in combination with excision to determine overall system efficiency (10% completion) We made minor construct revisions to and generated pAtUBQ10:GFP transgenic 717 hybrid poplars for future editing and isolated multiple transgenic events (rather than dsRed), which will serve as material for transformation of editing/excision constructs to test system efficiency. We have also cloned and prepared construct components to test excision on T-DNAs of different sizes, mimicking the presence of Cas9 and other important vector components using the same induction scheme. Testing of these components will occur concurrently with the GFP editing vectors. Aim 2: Production and genomic assessment of edited/excised poplars (40% completion) To produce edited and excised hybrid poplar plants with desirable traits, we transformed 717 hybrid poplars with CRISPR/Cas9 excision constructs targeting the LEAFY (LFY) gene as well as two GIBERELLIN INSENSITIVE (GAI) homologous genes to produce semi-dwarf and sterile edited poplars. These constructs included a pAtCSP3:Cre-GR excision component, GFP as a fluorescence marker, and a hygromycin resistance gene. The predicted footprint size after excision with this construct was 252bp. The results of the first construct are contained in the two presentations: ASPB 2021 and SIVB 2021(Found at the following links: https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/StraussASPB2021.pdf, https://people.forestry.oregonstate.edu/steve-strauss/sites/people.forestry.oregonstate.edu.steve-strauss/files/Strauss%20SIVB%202021%20v7.pdf). We were able to obtain a single lfy mutant, fully excised transgenic event from this transformation, which will be used in Aim 3 (Figure 2A). We transformed a second construct targeting the second GAI homologue with a similar strategy. We isolated 150 transgenic events, 8 of which had desirable combinations of mutations. Excision of these transgenic events with dexamethasone is ongoing, but we anticipate obtaining enough events to obtain excised lfy mutants to perform genomic analyses in Aim 3. A recent study (https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.17353)illustrated DNA methylation, particularly CHH methylation within the Cre spacer sequence may influence Cre-mediated excision outcomes in LoxP flanked T-DNAs. We have begun analyzing the methylation status of generated gai/lfy events to better understand the effect of DNA methylation on excision outcomes. Treatment with the anti-DNA methylation chemical 5-azacytidine restored GFP expression in silenced but unexcised events (Figure 2B). 5-azacytidine treatment during regeneration resulted in high explant mortality, and events treated with moderate levels of 5-azacytidine showed no improvement in excision rates (Figure 2C). As an alternative approach, we developed artificial microRNA constructs which target the native poplar DNA methylase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) for knockdown. Tests comparing Cre excision efficiency with or without the DRM2 knockdown element are ongoing. We also finished construction and began transformation of pAtUBQ10:Cre transgenics (expected to produce worst-case scenario recombinogenic toxicity) for analysis in Aim 3. Aim 3: (0% completion). We are in the process of generating all transgenic events and controls required to begin Aim 3 analyses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Goralogia, G.S., Redick, T.P. & Strauss, S.H. Gene editing in tree and clonal crops: progress and challenges. In Vitro Cell.Dev.Biol.-Plant 57, 683⿿699 (2021). https://doi.org/10.1007/s11627-021-10197-x
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