Progress 06/01/21 to 05/31/24
Outputs
Target Audience: The first target audience is the scientific community for grapevine and fruit tree genomic and breeding research. Any research output in grapevine can be applied to other clonally-propagated plant material. In that regard, we recently published a review of RNP delivery in fruit-tree models introducing the overall concept of RNP delivery via Cell-Penetrating Peptide(doi:10.3389/fpls.2022.979742.ecollection 2022). The second target audience is the National Grape Wine Industry, whose representatives have demonstrated interest in developing marker-free gene-edited grapevine material. The National Grape Research Alliance is a grape and wine industry consortium committed to promoting gene editing technologies that are part of their research priorities (https://graperesearch.org/research/). In September 2022, we coordinated a meeting to inform two NGRA representatives of our scientific progress. A last meeting is scheduled for October 2nd, 2024, to present the results and the project's next steps based on their feedback. The third target audience is the OSU community.The PI's department (Horticulture) consists of several PIs working on clonally-propagated models (hazelnut, ornamental trees, cherry, etc.) and are closely connected to their industry partners. A new faculty member in Plant Biotechnology was recently recruited. One aspect of research includes collaboration with several plant breeders in the Department whose models areclonal crops like hazelnut, ornamental trees, and others and for which DNA-free gene editing is a promising research avenue to develop new varieties associated with their trait of interest. The fourth target audience is the local and national wine industry. As a core faculty member of the Oregon Wine Research Institute, the Project leader and Co-Project Leader has reached out to the local wine industry with several educational materials to introduce new editing technologies and their tangible impact on wine production. Several presentations (oral and poster) were delivered to local conferences and technical meetings with growers. A progress report was released in April 2021to the Vine to Wine OWRI Newsletter (https://owri.oregonstate.edu/owri/vine-wine-newsletter), an online portal managed by the OWRI. A final newsletter is in process of being published in October 2024 Changes/Problems:Two major changes have been made in the experimental approaches. Firstly, to determine the cell penetrating efficiency among the CPPs, Cas9 protein complexed with CPP candidates was proposed to be used followed by the assessment of mutation rates.Instead of this approach, we constructed GFP-Cas9 protein which would allow for visual screening of embryogenic cells for the internalization of GFP-Cas9 protein. In addition, HRM analyses were used to assess the editing rate, which is proportional to the delivery efficiency of CPPs. Secondly, the negative zeta potential of the VirD2-Cas9 RNPs did not increase as much in conjugated CPP compared to that of complexed CPP at 1:100 molar ratio. The higher zeta potential of the CPP-RNP complexation through electrostatic forces imparts more endocytic activity to the complex through the plasma membrane. Under the reaction conditions, both complexation and conjugation through cysteine and maleimide are expected. So, the complexation of RNPs with CPP is being followed instead of making RNPs using CPP-conjugated VirD2-cas9 protein.? What opportunities for training and professional development has the project provided?As mentioned in the other Products section, the Post-Doctoral Research Associate Gouthu was trained in the technique of FACS at the Genomics & Cell Characterization Core Facility (University of Eugene). In addition, The Research Associate served as a mentor for one exchange visitor Student from France (Fabian Bustos - ENSAT - Toulouse - October 2022 - February 2023), who was trained in all the molecular biology techniques related to the majority of experiments designed to test the RNP activity, the in vivo and in vitro complexations tests to the RNP, the delivery conditions to microvine cells, the High-Resolution melting analyses. In June 2023, another Post-Doctoral researcher (Charlotte Song) in the Deluc Lab was recruited as part of a Non-Assisted Cooperative Agreement with Roger Thilmony's group who is a molecular biologist working with the microvine system (Crop Improvement and Genetics Research - Albany - CA). The Post-Doctoral research project is to test two geminivirus-derived viral vectors to deliver transiently a CRISPR-Cas9 cassette in microvine cells for transgene-free editing purposes. The primary delivery was with Agrobacterium-tumefaciens (EHA 105). In addition to the primary objectives,the Post-Doctoral Researcher Song was tasked to open her research approaches and to work with Research Associate Gouthu to get the proper training to test the delivery of the viral vector constructs with the CPPs as well. How have the results been disseminated to communities of interest?So far, the results have been disseminated through technical newsletters and oral and poster presentations to the National Grapevine Committee (See Other product section for details). As part of the project's outreach plan, an advisory committee, which included two researchers (Mickael Malnoy - Edmund Mach Institure - Italy; Gan-Yuan Zong - Research Leader - USDA ARS - Grape Genetics Research Unit)and two stakeholders and officers of the National Grape Research Alliance (Dan Martinez - Martinez Orchads - Treasurer of the NGRA, Nick Dokoozlian, Research Chair of the NGRA - E& J Gallo Winery), was informed of the progress of the work through annual meetings. The final one is being scheduled for October 2ndthis year. Additional dissemination will be allocated from the PL's discretionary funds to cover travel conference expenses for the Research Associate Gouthu in the following fiscal year. In addition, two other manuscripts from the research are scheduled: 1) One manuscript, which is in preparation, focuses on the use of CPP-mediated delivery of ssDNA-RNP for improved HDR before the end of the year 2024, with the expectation of demonstrating the plant regeneration from CPP-treated plants and, therefore, the Transgene-free applicability of the methodology. 2) A second manuscript focusing on screening CPPs for editing efficiency with RNP for rapid transgene-free knockout generation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Modern plant breeding can use genome editing to introduce desirable traits into clonal crops. Still, the transgenic nature of the edited material and the imperfect repair following the action of the CRISPR/Cas9 can pose a problem to commercializing gene-edited crops. The former issue stems from inefficient methodologies to deliver CRISPR/Cas9 as RiboNucleoProtein (RNP) to intact plant cells, which explains the current use of genetic transformation to express the Cas9 in the genome of the targeted crop. The latter problem is due to the low percentage of precise repair, as the repair template DNA may not be accessible at the double-stranded break site. The project addressed these two issues by establishing a transgene-free technique for precise editing using intact and regenerable grapevine embryogenic tissues. We aim to deliver CRISPR -RNP into intact regenerable microvine cells with the repair template tethered to it to increase the frequency for precise editing. Cell Penetrating Peptides (CPP), a class of short peptides, are proposed to facilitate the RNP complex's delivery across the cell wall and cell membrane. Establishing this gene-editing technique will accelerate the integration of innovative gene-editing tools into grapevine breeding programs. Besides, the approach can be versatile enough to be applied to other vegetatively propagated crops. Objective 1: We proposed screening six Cell Penetrating Peptide (CPP) candidates for their relative efficiency in delivering CRISPR reagents to intact cells. First, we constructed and synthesized a Cas9 protein fused with Green Fluorescent Protein (GFP) for rapid visual assessment through microscopy. The purified synthetic protein was complexed with the six CPP candidates and delivered to wild-type microvine embryogenic cells. Additionally, to assess the functionality of the RNP once internalized in the cells, we repeated the experiments with transgenic embryogenic lines expressing the GFP gene, using a regular Cas9 protein containing single-guide RNA targeting the GFP gene. Genomic DNA from the GFP-related calli was tested for mutational editing using High-Resolution Melting techniques (HRM). From the experiment using the fusion Cas9::GFP, many cells were found with GFP fluorescence, confirming the delivery across the cell wall. However, it was difficult to properly quantify the relative RNP-delivering efficiency among the CPPs due to the autofluorescence background of the plastids. By contrast, the HRM carried out on the second experiment showed a relatively higher editing rate of the GFP gene in the cells delivered with CPP1 (BP100(KH9)) and CPP2 ((BP100)2K8). Following these results, we created a fusion Cas9 protein (VirD2-Cas9) to ensure the tethering of the donor/repair DNA template near the RNP. Quality controls regarding the protein complex's functionality were carried out following the protein expression and its purification. This includes the Cas9 ability to cut DNA, the nicking activity of the VirD2 portion, and the covalent binding of single-stranded DNA donor template to VirD2. Further, the biochemical environment was optimized to prevent protein degradation and promote the complexation of the protein with the CPPs. Finally, the overall protein complex size and charge in the presence of CPPs were evaluated using a nanoparticle size/surface properties measuring instrumentation. The gel retard experiments confirmed a better complexing capacity of CPP1 and 2 to VirD2-Cas9 compared to the other CPPs. This could explain the higher editing rate observed in the cells using these CPPs depending on their higher cellular delivery. In vitrotests also showed that over 50% of VirD2-Cas9 proteins were bound to the donor template. The overall charge of the complex, an essential feature for optimized cellular entry of RNPs across cell walls and membranes increased after CPP complexation. Still, no significant change in the size was observed. Objective 2: A donor/repair DNA template (ssDNA) with targeted modifications was designed to convert GFP into a Blue Fluorescent Protein gene (BFP). The DNA donor template contains a base substitution of T>C at the 196thposition of the GFP gene, which will substitute the 65th amino acid tyrosine to histidine, and a base substitution at the 194thposition to eliminate the PAM recognition sequence to prevent further editing. The donor template also has a 25 bp repeat sequence that facilitates its binding to the VirD2 portion of the chimeric Cas9 protein, and it is chemically modified on one end to protect it from cellular degradation. Two single-guide RNA (sgRNA) were designed to cover the 196thbase of intended T>C substitution in the GFP gene andin vitrosynthesized. The preparation for cellular delivery of the RiboNucleoProtein (RNP) complexes with the CPP was performed in three steps. First, sgRNA and VirD2-Cas9 protein were assembled, resulting in the formation of a negatively charged RNP. Secondly, covalent attachment of ssDNA to the RNP was performed. The ssDNA-RNP endonuclease activity was confirmed throughin vitrocleavage assays on a template plasmid containing the GFP gene. Finally, the aliquots of the assembled ssDNA-RNP were complexed with individual CPPs at the molar ratio of 1:50. Each CPP-ssDNA-RNP was delivered to ~200 mg of fresh microvine calli sub-cultured for two weeks. The CPP-complexed RNPs were added to the calli, followed by a vacuum cycle to help the adsorption of RNP complexes to the cell wall. Calli were maintained for four days in an appropriate medium before being harvested for downstream applications. Appropriate controls were set to help interpret the results from treatments, including 1) RNP alone, 2) RNP-CPP without ssDNA, and 3) RNP and ssDNA complexed separately. Objective 3: Fluorescence-activated cell Sorting (FACS) assays were not successful in proving the precise editing of GFP to BFP due to limitations in the purity of cell preparations. So, (AmpliSeq) we used the Illumina-based Amplicon-sequencing technique, which allows a more accurate and quantitative way to identify and estimate the editing rate. Genomic DNA extracted from the treated calli was used to generate a 300 bp amplicon straddling the expected editing site; the purified PCR products were sent then for AmpliSeq analyses. The resulting data were analyzed using Geneious Prime bioinformatic software to merge 150 to 230,000 reads per sample covering the targeted region of the GFP gene. Only 0.15% precise repair was observed when Cas9 RNP and ssDNA complexed to CPP1 were delivered to the cells. In this control treatment, the ssDNA cannot attach to Cas9 RNPs in the cells. The availability of ssDNA at the double-stranded break to act as a template for precise repair is left to chance. However, the HDR editing rate was 10.3% when delivered with VirD2Ca9 RNPs and ssDNA complex to CPP1, which ensures the proximity of the DNA donor template to the RNP. When only VirD2Cas9 RNPs were delivered with CPP1 without any ssDNA, only unprecise editing can happen, but surprisingly, it was as low as 0.25%, indicating a poor cellular delivery. Therefore, while CPP1 was an efficient vehicle to deliver to intact cells the chimeric Cas9 (VirD2::Cas9), the RNP delivery is better when using covalently bound ssDNA, probably due to the additional negative charge brought to the overall RNP complex. Overall, our experimental results addressed two aspects of our research project. Cell-Penetrating peptides could effectively deliver a CRISPR RNP to cells under specific conditions. Maintaining the DNA repair template near the VirD2-Cas9 can significantly increase the precise editing rate and improve cellular delivery. We are currently repeating the experiment to generate individual plantlets. If successful, this approach will be an alternative strategy to the currently protoplast-based approach for transgene-free gene editing.
Publications
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience: The first target audience is the scientific community for grapevineandfruit tree genomic and breeding research. Any research outcome validated through this project, which uses grapevine can be applied to other clonally-propagated plant material. In that regard, we recently published areview ofRNP delivery in fruit-tree models (doi:10.3389/fpls.2022.979742.ecollection 2022). We introduced the overall concept of RNP delivery via Cell-Penetrating Peptide. The second target audience is the National Grape Wine Industry, which representatives have demonstrated interest in the development marker-free gene-edited grapevine material. The National Grape Research Alliance, which is a consortium of grape and wine industry stakeholders is also committed to promoting gene editing technologies that are part of their research priorities (https://graperesearch.org/research/). In September 2022, we coordinate a meeting to informtwo NGRA representatives of our scientific progress. The third and fourth target audience is the OSU community and the local wine industry. The PI's department (Horticulture) consists of several PIs working on clonally-propagated models (hazelnut, ornamental trees, cherry, etc.) and are closely connected to their industry partners. A new faculty position in plant Biotechnology including gene editing technology was recently accepted at OSU.Any research outcome from this project could lead in the near future will lead topotential collaborations with the new faculty member. The local wine industry is finally reached for educational purpose to introduce new editing technologies and their tangible impact on wine production. The project leader is also a core Faculty of the Oregon Wine Industry Institute, which releases every month outreach documentations. A progress report will be released by December 2023 to theVine to Wine OWRI Newsletter (https://owri.oregonstate.edu/owri/vine-wine-newsletter). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?It will be disseminated by December this year 2023 with the Oregon Wine Research Institute. What do you plan to do during the next reporting period to accomplish the goals?CPP-RNP's deliveryto convert eGFP into BFP-expressing microvine cells is the final experiment to be conducted and it will be done within a few weeks now. Two major approaches will be carried out to assess the HDR-mediated editing efficiency on treated microvine embryogenic cells that are 1) Fluorescence Activated Cell Sorting (FACS) to quantify the numbers of cells and 2) Next-generation sequencing analyses. While the NGS will be performed on intact embryogenic cells, the FACS will be conducted on protoplasts that will be generated soon after the incubation of the eGFP-expressing embryogenic cells with the CPP-mediated RNP. Since our request to extend the award at no cost was granted, and if we succeed in increasing the editing rate, we will explore the opportunity to generate individual plants expressing the BFP to confirm that is possible to generate marker-genome editing until plant regeneration.
Impacts
What was accomplished under these goals?
The project's main goal is to establish a new scientific approach for transgene-free gene editing through Homology Directed Repair (HDR) using intact, regenerable embryogenic tissues in grapevine. The primary research objective is to deliver CRISPR Ribonucleoproteins (RNPs) along with a tethered repair template to increase the HDR event frequency into intact regenerable cells of microvine. Complexing or conjugatingCell Penetrating Peptides (CPP) to the gene editing reagents (RNP) is proposed for the delivery of this RNP complex across the cell wall and cell membrane. Establishing this gene-editing technique will give an advantage to the research community to speed up the process of integrating gene-editing technology into grapevine breeding programs. The major scientific tasks and objectives were completed for the first objective. To screen the CPP candidates for cell penetration efficiency and deliver CRISPR reagents across cell walls, GFP-Cas9 fusion protein has been constructed. Purified GFP-Cas9 protein was complexed with the five CPP candidates and delivered microvine embryogenic cells. The calli were screened for RNP internalization in the cells, it was not possible to accurately quantify the relative RNP-delivering efficiency among the CPPs. However, High-Resolution Melting Curve (HRM) analysis after the delivery of RNPs to edit the GFP gene in embryogenic callus showed CPP2 ((BP100)2K8) and CPP1 (BP100(KH9)) with better delivery efficiency. To prepare for the delivery of CRISPR RNPs with tethered single-stranded donor template to improve HDR efficiency, VirD2-Cas9 fusion protein has been purified. Quality controls to assess the proposed functionalities of the fusion protein including, A) Endonuclease activity of VirD2-CAs9 fusion protein on the target GFP gene, B) NIcking activity of VirD2 on T-plasmid containing RB-recognition sequence, and 3) Covalent binding of single-stranded donor template to VirD2-Cas9 fusion protein at equimolar concentrations of DNA have been confirmed in vitro. We achieved over 50% of VirD2-CAs9 protein bound to the donor template. The optimum buffer system and reaction conditions for the complexing of CPP to CRISPR RNPs have been optimized. Complexing of CPP-RNPs is confirmed through semi-denaturing SDS-PAGE analysis. Particle size and zeta potential of the RNP-CPP complex were assessed through Dynamic Light Scattering (DLS) analysis. The zeta potential, which is important for cellular entry of RNPs increased after CPP complexation, but no significant change in particle size was observed. Major activities for the second objective were completed: The donor template with targeted modifications has been designed and microvine embryogenic callus expressing GFP has been generated. The donor template has been designed with a base substitution of T>C at 196th position of GFP, which will substitute the 65th amino acid tyrosine to histidine, and a base substitution at the 194th position to eliminate the PAM recognition sequence. The donor template is appended with a 25 bp right border sequence of T-plasmid that attaches to the VirD2 protein. The single-stranded donor template was synthesized with phosphorothioate-modified ends to protect it from nucleases. Experiments to optimize the binding of RNP and donor template were undertaken and binding of donor template to RNP through RB-recognition sequence has been confirmed (over 50%). Microvine embryogenic calli have been generated from anther cultures expressing GFP to use for gene editing experiments. The cultures are being maintained and their embryogenic potential has been confirmed. For the third objective, the CPP-RNP complexes are readied following the optimized method for delivery into embryogenic cells. To assess the initial editing rate and improvement of HDR rate, we are assessing the use of fluorescence cell sorter using protoplasts prepared from the CPP-RNP treated cells and we are successful in preparing high-purity protoplasts.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Transgene-free genome editing and RNAi ectopic application in fruit trees: Potential and limitations
Satyanaryanana Gouthu, Christian Mandelli, Britt A Eubanks, Laurent G Deluc Frontiers in Plant Sciences 2022 Oct17;13:979742. doi: 10.3389/fpls.2022.979742.ecollection 2022.
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience: The first target audience is the scientific community forgrapevine and fruit tree genomic and breeding research. While the research model is the grapevine, any research outcome revealed through this project can be applied to any clonally-propagated plant material. Thus, the dissemination of the results would go beyond the grapevine model. In that regard, we recently submitted a review to a fruit-tree-relatedresearch topic at Frontiers in Plant Sciences related to RNP delivery. Although, we did not explicitly provide any results regarding the current project we introduced the overall concept of RNP delivery via Cell-Penetrating Peptide. The second target audience is the national Grape Wine Industry which is interested in the development of any technology that is transgene-free for genetic improvement.The National Grape Research Alliance, which is a consortium of stakeholders of the grape and Wine Industry,is committed to promoting gene editing technologies that are part of their research priorities (https://graperesearch.org/research/). The third and fourthtarget audience is the OSU community and the local wine industry. The PI's department (Horticulture) consists of several PIs working on clonally-propagated models (hazelnut, ornamental trees, cherry, etc.) and areclosely connected to their industry partners. Any research outcome from this projectcould lead in thenear future topotential applications withother cropsin Oregon. The local wine industry is finally reached for educational purposeto introduce new editing technologies and their tangible impact on wine production. Changes/Problems:Two significant changes have been made in the experimental approaches. Firstly, to determine the cell-penetrating efficiency of CPPs carrying the chimeric Cas9 RNPs, Cas9 protein was proposed, followed by the assessment of mutation rates using different CPPs.But, the size of the cargo protein attached to CPP, such as 165 KDa Cas9 while screening versus 215 KDa VirD2-Cas9 while performing the HDR-mediated editing experiment. The size of GFP-Cas9 protein, 195 KDa, is much closer to VirD2-Cas9 protein and further allows for visual screening of embryogenic cells to internalize GFP-Cas9 protein. Secondly, the negative zeta potential of the VirD2-Cas9 RNPs did not increase as much in conjugated CPP compared to that of complexed CPP at a 1:100 molar ratio. The higher zeta potential of the complex, formed by electrostatic complexing of cationic CPPs to negatively charged VirD2-Cas9 RNPs, imparts more endocytic activity through the plasma membrane. Under the reaction conditions, complexation through sgRNA-derived localized negative charge and conjugation through C-terminal cysteine and maleimide group of CPP is expected. So, the complexation of RNPs with CPP is being followed instead of making RNPs using CPP-conjugated VirD2-cas9 protein. What opportunities for training and professional development has the project provided?Training activities: As part of the research project, a senior Faculty Research Assistant was recruited in February 2022 to assist the CoPI Satyanarayana Gouthu in his work. The trained personlearned techniques relatedto 1) subculturing embryogenic cells and 2) production of the synthetic protein Cas9-VirD2 and Cas9-GFP. The CoPI Gouthu performed the training. Professional Development: The PI Deluc gave a seminar at the Department of Horticulture on May 7th, 2022, to reach out to the OSU community (student and faculty) regarding the project. The PI Deluc also gave a "Gene Editing Technology" presentation atthe Grape Day event (April 26th, 2022 - https://owri.oregonstate.edu/owri/events/grape-day)organized by the Oregon Wine Research Institute every year. The presentation included anintroduction of thecurrent project. The Co-PI Gouthu provided a poster Presentation on CPP-delivery at the International Conference of Grapevine Physiology and Biotechnology sponsored by the International Society of Horticulturethat was taking place in South Africa (Remote event - https://www.ishs.org/symposium/529) from October 31 to November 5th 2022. How have the results been disseminated to communities of interest?While the project's scope has been communicated, the results have not yet been disseminated to any communities yet.The announcement award was communicated in August 2021 through the Vine to Wine newsletter of the Oregon Wine Research Institute (https://owri.oregonstate.edu). What do you plan to do during the next reporting period to accomplish the goals?Besides the proposed changes in the approach of the first objectiveand described in the Changes/problem section of the progress report module, the rest of the project will be accomplished as mentioned in the agency-approved application and according to the REEportguideline (Page 46)
Impacts
What was accomplished under these goals?
The project aims to develop an approach for transgene-free gene editing through Homology Directed Repair (HDR) in grapevine. Because commonly adapted gene editing techniques either generate transgene-free edited plants in T1 generation or require protoplast regeneration, they cannot be applied in clonally propagated crops such as grapevine. So, the project attempts to deliver CRISPR gene editing reagents along with a repair template into microvine intact regenerable cells using Cell-Penetrating Peptides (CPP) for delivery across the cell wall and cell membrane. Establishing such a gene editing technique through a transgene-free approach will give an advantage to the research community to speed up the process of integrating gene editing into grapevine breeding programs without being labeled GMO since there will not be any "Foreign DNA. Furthermore, this technology ofeditedgrapevine materialwill benefit the grape industry by generating improvedgene-editedgrapevine genetic resources. So far,for the first objective, to enable the screening of CPPs for cell-penetrating efficiency and internalization of CRISPR reagents, the GFP gene has been cloned N-terminus of Cas9 to express GFP-Cas9 fusion protein. Purified GFP-Cas9-Cys protein was complexed and conjugated with the five CPP candidates and delivered to microvine embryogenic cells. The callus was screened for GFP-Cas9 internalization in the cells. To deliver the CRISPR RiboNucleopProteins (RNP) along with tethered donor templates with targeted modifications, the VirD2 gene from Agrobacterium has been cloned as the N-terminal fusion of Cas9. VirD2-Cas9 protein has been expressed and purified, and the endonuclease activity of purified chimeric protein on the target GFP gene has been confirmed in vitro. Covalent binding of a single-stranded donor template with VirD2-Cas9 fusion protein at equimolar concentrations of DNA and protein has been confirmed in vitro. Cas9 protein alone did not show any gel retardation; only VirD2-Cas9 showed. For the second objective, the donor template with targeted modifications has been designed, and microvine embryogenic callus has been generated. Abase substitution of T>C at 196thposition of GFP, which will substitute 65thamino acid tyrosine to histidine, and a base substitution at the 194thposition to eliminate the PAM recognition sequence, were implemented in thedonor template.The donor template is appended with a 25 bp Right Border sequence of Ti-plasmid that attaches to the VirD2 protein. The single stranded donor template was synthesized with phosphorothioate modified ends to protect from nucleases. To use for gene editing, embryogenic callus has been generated from anther cultures of the microvine expressing GFP. The cultures are being maintained on maintenance media and their embryogenic potential has been confirmed.For the third objective, the CPP-RNP complexes are being prepared for delivery into the embryogenic cellsto analyze the HDR rate through NGS analysis. For transgene-free gene editing in grapevine and most clonal crops, techniques to deliver CRISPR RNPs through the cell wall is required in order to regenerate the gene edited cells. The results indicate that CRISPR reagents linked to cationic CPPs can penetrate through the cell wall and cell membrane. The results also confirm that the DNA repair template can be tethered to RNP complex by using a chimeric VirD2-Cas9 to maintain the repair template's proximity to double strand break site and enhance the HDR rate. Further, zeta potential of the CRISPR RNP is increased several folds by complexing the with cationic CPP thereby enhancing the RNP-cell membrane interaction for cellular entry.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
"Transgene-free genome editing and RNAi ectopic application in fruit trees: Potential and limitations"
Invitation to submit a review by the editors of the Research Topic " Functional Genomics in Fruit Trees: from Omics to Sustainable Biotechnologies, Volume II" in Frontiers in Plant Science - Section Plant Breeding
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