Progress 09/01/20 to 08/31/24
Outputs
Target Audience:Results from this project were targeted to the scientific community through presentations at the annual ECDRE directors meeting as well as national and international plant science meetings. Additional outreach was through the submission and publication of journal publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities in citrus tissue culture, gene promoter characterization, gene editing development and gene analysis were provided for for four undergraduate students and three postdoctoral students. How have the results been disseminated to communities of interest?Results from these studies have been published or are being prepared for publication in peer-reviewed journals. Corresponding gene sets have been deposited in the NCBI BioProject. Project findings have also been presented at several meetings including the International Institute of Tropical Agriculture (IITA), CGIAR,Nairobi, Kenya;the Plant Genome Stability and Change-EMBO Workshop,Olomouc, Czech Republic;the Plant Center Spring Symposium, University of Georgia; and the Innovative Genomics Institute, UC-Berkeley. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Development of multiplexed gene editing and gene activation methods in citrus. Task 1. Establish citrus protoplast transformation systems. A stable callus maintenance, protoplast and transformation system was established. In summary, liquid culture conditions for Hamlin 89 (H89), Hamlin 19 (H19), Valencia 17 (V17) and Pineapple 17 (Pa17) embryogenic callus lines were determined. Protoplast isolation and gene transfection systems were subsequently developed and optimized, producing a protoplast transfection efficiency of approximately 26%. The establishment of these systems represent a first step in developing a transgene free CRISPR-CAS12a citrus protoplast transformation system. Task 2, Develop multiplexed Cas12a system for promoter editing. A transgene free CRISPR-CAS12a ribonucleoprotein (RNP) mediated mutagenesis system was established. Specifically, the wild type Cas12a enzyme with corresponding crRNA scaffolds were expressed and pre-assembled as a RNP complex. This RNP complex was engineered to target the citrus CsPH5 gene as a marker to measure gene editing efficiency in transfected protoplasts. Result showed that the target CsHP5 site was successfully mutated by the CRISPR-Cas12a based RNP with a 90% editing efficiency, indicating the CRISPR-Cas12a based RNP delivery system provides a powerful tool for citrus genome editing. One obstacle encountered under this objective was the inefficiency of regenerating plants from edited citrus protoplasts. To address this issue an RNP system for CRISPR-Combo, a new gene editing system that both directs targeted mutations and activates specific gene transcripts was developed. The goal of this approach was to use CRISPR-Combo to activate citrus morphogenic genes as means to enhance the ability of gene edited citrus protoplasts or transformed tissue to regenerate into whole plants. Findings from this study are listed below in Objective 3, task 2. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming. Task 1. In silico identification of target gene promoter sequences. Meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars identified a set of defense associated genes that were significantly upregulated in resistant and tolerant cultivars but not in sensitive cultivars. In addition, we utilized the citrus tristeza virus (CTV) vector to express a tagged ribosomal protein, hfRPL18. This system allows for the selective isolation of mRNAs associated with ribosomes carrying the hfRPL18 protein. The CTV vector replicates and expresses the hfRPL18 protein in the phloem, making it possible to capture phloem specific ribosomes. mRNAs associated with these ribosomes likely represent transcripts specific to the vascular phloem. Results from these studies identified over 400 putative phloem expressed citrus genes. The combined identification of these phloem associated genes provides a unique set of targets for gene editing efforts aimed at manipulating defense and environmental phloem responses within citrus. Task 2. Expression analysis and validation of identified target genes. To confirm phloem and tissue specific expression of identified citrus promoter sequences a GUS-based marker expression system was used within an Arabidopsis plant system. Continuing studies have validated multiple citrus phloem promoter sequences as well as several constitutive leaf tissue promoters. Combined these studies have identified tissue specific promoter sequences, providing a set of unique tools for future efforts aimed at manipulating defense, developmental and agronomic traits in citrus. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. QPCR analysis of promoter edited protoplasts and calli for regenerated germplasm. The CRISPR-Cas12a ribonucleoprotein (RNP) system was established in citrus protoplasts with a 90% gene editing efficiency. Initial efforts focused on establishing embryogenesis and embryo germination protocols to promote the regeneration of edited citrus protoplasts to embryos and ultimately rooted plantlets. While systems were established to promote embryo to plant development the conversion of citrus protoplasts to embryos was not successful. To address this obstacle, transgene CRISPR systems were also developed and optimized for citrus hypocotyl stem sections and hairy roots. Combined these systems provide a robust means to investigate CRISPR applications in citrus, particularly toward the use of CRISPR-Combo for the activation of morphogenic genes to improve citrus regeneration. Task 2 Regeneration and in situ expression analysis of promoter edited earn and germplasm. To address inconsistencies in citrus regeneration a CRISPR-Combo system capable gene editing and targeted gene activation was employed to enhance the expression of known plant morphogenic genes. The hypothesis being that increased morphogenic gene activity would enhance citrus plant regeneration. Using this approach twenty different CRISPR-Combo transformation constructs targeting ten different morphogenic genes and two CLas associated genes, CsNPR3-HLB defense and CsLOB1-HLB susceptibility, were created and tested for their impact on citrus regeneration and target gene editing. For these studies a Carrizo citrange hypocotyl transformation system was assessed for the ability of CRISPR-Combo constructs that targeted different morphogenic genes to enhance shoot regeneration. Higher regeneration rates, characterized by the emergence of dense clustered shoots were observed upon activation of morphogenic genes CsSERK1L, CsWUS3, and CsBBM3. A harry root transformation system using A. rhizogenes was also employed to assess the impact of these CRISPR-Combo editing constructs in citrus regeneration. Within this system four constructs targeting the disruption of CsNPR3 and the activation of morphogenic genes CsBBM3, CsIPT, CsSERK1L and CsSTM displayed significant increases in the number of harry root transformations in comparison to control constructs. PCR amplification and Sanger sequencing confirm the gRNA directed disruption of targeted CsNPR3 genes. Interestingly, CRISPR-Combo constructs activating these same morphogenic genes but targeting the CsLOB1-HLB susceptibility gene failed to enhance harry root transformations. This suggests that CsLOB1 involvement in growth and development also affects plant regeneration. Combined these findings demonstrate the usefulness of multiplexed gene editing systems to simultaneously enhance plant regeneration and target gene disruption.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Tuncel, A., Pan, C., Sprink, T. et al. Genome-edited foods. Nat Rev Bioeng 1, 799816 (2023).
- Type:
Other
Status:
Submitted
Year Published:
2024
Citation:
Culver, J.N., Vallar, M., Burchard, E., Kamens, S., Lair, S., Qi, Y., Collum, T.D., Dardick, C., El-Mohtar, C-A., Rogers, E.E., 2024. Citrus Phloem Specific Transcriptional Profiling Through the Development of a Citrus Tristeza Virus Expressed Translating Ribosome Affinity Purification System. Submitted.
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Results from this project have been accomplished through journal publications as listed under products. Changes/Problems:COVID restrictions, including hiring and space restrictions significantly delayed the onboarding of key personnel for this project thus slowing our progress. In addition, the recalcitrant nature of citrus to tissue culture regeneration required us to develop and apply new CRISPR editing and activation systems to promote and improve plant regeneration systems needed for this project. Because of these delays we are requesting a 2nd no cost extension of 12 months. With the new systems for enhanced citrus regeneration and the needed personnel in place we anticipate completing the project within the requested extension. What opportunities for training and professional development has the project provided?Training opportunities in citrus tissue culture, gene editing development and gene analysis were provided for one graduate student and two postdoctoral students. How have the results been disseminated to communities of interest?Results have been disseminated primarily through the publications listed. What do you plan to do during the next reporting period to accomplish the goals?As outlined, using a new CRISPR-Combo system we have achieved a significant advancement in efforts to enhance citrus regeneration. The requested 2nd NCE will allow us to further apply this system to complete efforts to modify the phloem expression of citrus genes associated with HLB resistance and/or susceptibility. Our initial CRISPR-Combo morphogenic gene activation studies have focused on Carrizo citrange due to its amenability to regeneration. With the additional requested 2nd NCE, efforts will examine more recalcitrant yet economically important citrus varieties including sweet orange varieties Hamlin and Valencia. Specific studies will investigate the use of morphogenic gene activation to enhance plant regeneration in edited protoplasts, embryogenic callus and hypocotyl sections from designated sweet orange varieties as outlined in Objective 1. Efforts will also investigate the application of the CRISPR-Combo system to introduce gene modifications that suppress HLB as proposed in Objectives 2 and 3. Additional, efforts will explore the previously established embryogenic protoplast and calli systems for morphogenic gene activation and editing using the developed CRISPR-Combo ribonucleoprotein system (RNP). RNP deliver will occur via PEG or liposomes to protoplasts and through biolistic delivery to the shoot apical meristems and hypocotyl cell layers of targeted sweet orange varieties. We anticipate that outcomes from the requested 2nd NCE will result in the development of powerful CRISPR-Cas systems for enhanced citrus regeneration and phloem targeted gene-editing. The application of this system is critical to achieving the proposed genome modifications to citrus germplasm in an efficient and routine manner for subsequent testing against CLas infection.
Impacts
What was accomplished under these goals?
Objective 1. Development of multiplexed gene editing and gene activation methods in citrus. Task 1. Establish citrus protoplast transformation systems. A stable callus maintenance, protoplast and transformation system was established as reported in previous progress reports. Task 2, Develop multiplexed Cas12a system for promoter editing. CRISPR-CAS12a ribonucleoprotein (RNP) mediated mutagenesis has been established as reported previously. In addition, we developed an RNP system for CRISPR-Combo, a new gene editing system that can both direct targeted mutations and activate specific gene transcripts. CRISPR-Combo was used to investigate the activation of citrus morphogenic genes to enhance the regeneration of gene edited cells, which is a major obstacle in citrus transformation. Twenty different CRISPR-Combo systems targeting ten different morphogenic genes and two CLas associated genes, CsNPR3 and CsLOB1, were created and tested for their impact on citrus regeneration and target gene editing. Results from this study revealed significant enhancements to citrus culture regeneration and are outlined below in Objective 3, task 2. Future efforts will explore the previously established embryogenic protoplast and calli systems for morphogenic gene activation and editing using non-transgene CRISPR-Combo RNPs. RNP deliver will occur via PEG or liposomes delivery to protoplasts and through biolistic delivery to the shoot apical meristems and hypocotyl thin cell layers of targeted sweet orange varieties. Task 3, Develop CRISPR-Act3,0 for tissue-specific activation of target genes in phloem. CRISPR-Act3.0 enhancer constructs targeting phloem gene specific promoters identified in Objective 2 are currently being tested within an Arabidopsis model system for phloem activation. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming. Task 1. In silico identification of target gene promoter sequences. As previously reported, in silico and meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars were performed, identifying gene transcripts that are significantly altered in response to HLB. This information will be used in future CRISPR-Combo constructs that will target the transcriptional activation of both morphogenic genes for enhanced plant regeneration as well as the identified HLB response genes. Task 2. Expression analysis and validation of identified target genes. As stated above we are applying the CRISPR-Combo system as a means to enhance citrus regeneration from culture. The enhanced regeneration observed with this system has the potential to overcome the recalcitrance of citrus to regenerate and allow for the screening of edited target genes as proposed. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. qPCR analysis of promoter edited protoplasts and cam for regenerated germplasm. Gene editing directed by a CRISPR-Cas12a ribonucleoprotein (RNP) system as well as a transgene directed CRISPR-Combo system have been established within citrus protoplasts and hypocotyl stem sections. In addition, an RNP system for the CRISPR Act3.0 activation system was also validated in vitro for assembly and gene editing activation. Task 2 Regeneration and in situ expression analysis of promoter edited earn and germplasm. The major goal of this project is to develop gene-editing systems that promote the modified expression of phloem expressed target genes associated with defense against and or susceptibility to Clas. While we have made significant progress on developing multiplexed gene editing systems for this study and identifying phloem specific genes for targeted gene editing a significant obstacle has been the inability to regenerate citrus from transformed protoplasts or callus tissues as many citrus cultivars are recalcitrant to regeneration efforts. To address this bottleneck, we incorporated a newly developed CRISPR system, termed CRISPR-Combo, that can direct both targeted gene editing and the activation of morphogenic genes that enhance plant regeneration. Using this approach twenty different CRISPR-Combo systems targeting ten different morphogenic genes and two Clas associated genes, CsNPR3-defense and CsLOB1-susceptibility, were created and tested for their impact on citrus regeneration and target gene editing. For these studies Carrizo citrange (Carrizo citrange trifoliate hybrid, Citrus sinensis "Washington" sweet orange X Poncirus trifoliata) was selected for CRISPR-Combo testing using Agrobacterium-mediated stable transformation. The regeneration rate of Citrus explants was assessed and compared among various CRISPR-Combo constructs that targeted different morphogenic genes. Significantly higher regeneration rates, characterized by the emergence of dense clustered shoots were observed upon activation of morphogenic genes CsSERK1L, CsWUS3, and CsBBM3. PCR amplification and Sanger sequencing were subsequently used to confirm the gRNA directed disruption of targeted Clas associated genes. These findings demonstrate the potential of the CRISPR-Combo system to overcome citrus associated regeneration bottlenecks. One drawback of the enhanced shoot proliferation associated with the CRISPR-Combo activation of target morphogenic genes was that the majority of the regenerated shoots were abnormal, did not produce roots and were not suitable for micrografting onto rootstock material. To address this issue modifications were made to the culture media, ultimately removing any growth hormones that are normally included to promote plant regeneration. On media lacking growth hormone normal shoot development was observed on hypocotyl sections transformed with CRISPR-Combo targeting CsSERK1L morphogenic gene activation while controls fail to develop any shoots. These results demonstrate that the activation of targeted morphogenic genes in the absence of additional growth hormones is sufficient to produce normal shoot production suitable for rooting and/or micrografting. Combined these results represent a substantial improvement in the ability to apply gene editing tools to improve citrus germplasm.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Fang, H., Culver, J.N., Niedz, R.P., Qi, Y., 2023. Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line. Methods Mol Biol 2653, 153-171.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kavuri, N.R., Ramasamy, M., Qi, Y., Mandadi, K., 2022. Applications of CRISPR/Cas13-Based RNA Editing in Plants. Cells 11.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Pan, C., Li, G., Bandyopadhyay, A., Qi, Y., 2023. Guide RNA library-based CRISPR screens in plants: opportunities and challenges. Curr Opin Biotechnol 79, 102883.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Zhang, L., Li, G., Zhang, Y., Cheng, Y., Roberts, N., Glenn, S.E., DeZwaan-McCabe, D., Rube, H.T., Manthey, J., Coleman, G., Vakulskas, C.A., Qi, Y., 2023. Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants. Genome Biol 24, 102.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Results from this project have been presented at the annual ECDRE directors meeting. Additional outreach has been through the submission and publication of journal outlines as listed under products. Changes/Problems: COVID19 restrictions, including hiring and space restrictionsslowed some experiments anddelayed the hiring of some project personnel. Due to these issues we have requested a no cost extension of one year. What opportunities for training and professional development has the project provided? Training opportunities in citrus tissue culture and gene analysis were providied for two postdoctoral students. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We have established citrus protoplasts and CRISPR-RNP gene editing systems. Additional studies will continue developing CRISPR tools for tissue (phloem) and pathogen response specific gene editing. In addition, as noted above one main bottleneck has been thetime and efficiency associated with regenerating transformed citrus cells. Through on going and planned experiments we are adapting the CRISP-Combo system to citrus for both targeted gene editing and enahnced plant regeneration. This will involve the screening of ~20 putative morphogenic genes for assemsement of their impact on citrus regeneration.
Impacts
What was accomplished under these goals?
Objective 1. Development of multiplexed gene editing and gene activation methods in citrus. Task 1. Establish citrus protoplast transformation systems.A stable callus maintenance procedure including both solid plate culture and liquid suspension culture has been developed. Citrus protoplast isolation and polyethylene glycol (PEG) mediated protoplast transfection has been established. Citrus cells from embryogenic callus cultures were used to produce protoplasts via standard isolation methods. Reporter constructs carrying the marker genes were subsequently used to test PEG mediated protoplast transfection. The average transfection efficiency was determined to be 30-90% depending on the construct used. Parameters such as protoplast numbers, target DNA concentration and incubation time of the transfection were subsequently optimized in order to promote greater transformation efficiency. Task 2. Develop multiplexed Cas12a system for promoter editing.?CRISPR-CAS12a ribonucleoprotein (RNP) mediated mutagenesishas been established. A CRISPR-Cas12a based RNP delivery system has been tested on the protoplasts derived from citrus callus cultures. The wild type Cas12a enzyme (LbCas12a) and the crRNA scaffolds based on the CRISPR-LbCpf1 system were designed to target specific citrus genes. These components were pre-assembled with a molar ratio of 1:2 to form the RNP complex.TheRNP complex was delivered to citrus protoplast through PEG mediated protoplast transfection developed in Task 1 above.Afterthe transfection, protoplasts were incubated in the dark for 48 hr followed by genomic DNA isolation and restriction fragment length polymorphism (RFLP) assay. Different concentrations (0.1, 0.01 and 0.001 μM) of RNP complex were tested in the targeted mutagenesis of citrus protoplast. The result shows that the target site has been successfully mutated by the CRISPR-Cas12a based RNP delivery system with a 0.1 μM concentration of RNP displaying a nearly 90% editing efficiency, which indicates the CRISPR-Cas12a based RNP delivery system is a powerful tool for citrus genome editing. In addition, we have developed an RNP system for CRISPR-Combo, a new gene editing system that can both direct targeted mutations and activate specific gene transcripts. In particular we are interested in activating specific citrus morphogenic genes to improve the rate of plant regeneration. CRISPR-Combo assembly requires polypeptides for the Cas nuclease, SunTag amplification scaffold and transcriptional activation (TAD) as well as gRNAs targeting gene activation and editing. For these studies, we established a recombinant CRISPR-Combo protein expression system for the Cas nuclease, SunTag, and TAD polypeptides using the pET27b (+) vector and His-tag purification system per the manufacturer's protocol. Associated gRNAs are designed and synthesized for insertion into T7 transcription cassettes. These gRNA cassettes can be used for in vitro T7 RNA transcription and purification using commercial kits. Purified Cas, SunTag, TAD and gRNAs are mixed in an optimal ratio of 1:6:100:2 and incubated for 1 hour in 1X NEBuffer 3.1 at room temperature prior to testing. Initial in vitro testing has used a plasmid vector harboring a gRNA targeted restriction enzyme sequence with associated PAM site. Results have demonstrated the assembly and activity of this new RNP system. Ongoing studies are aimed at optimizing this system and moving it into citrus cells to examine its impact on plant regeneration, see Objective 3, task 2 below. Task 3. Develop CRISPR-Act3.0 for tissue-specific activation in phloem.CRISPR-Act3.0 enhancer constructs targeting citrus phloem gene specific promoters identified in Objective 2 are currently being developed.This system uses a defective dCas9 enzyme to target transcriptional activator domains to gRNA specified promoter sequences. Furthermore, additional phloem specific promoters have beenmoved into the CRISPR-Act3.0 T-DNA vectors so as to drive dCas9 expression in a phloem specific manner. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming. Task 1.In silicoidentification of target gene promoter sequences. Meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars were performed to identify gene transcripts that are significantly altered in response to HLB. This analysis has identified a set of defense associated genes that are significantly upregulated in resistant and tolerant cultivars but not in sensitive cultivars. This information is being used to generate phloem expressed CRISPR-Act3.0 constructs targeted at increasing the expression levels of select candidate genes in the phloem of HLB sensitive cultivars. Task 2. Expression analysis and validation of identified target genes.As stated above in Objective 1, Task 3, identified phloem specific promoters are being investigated as a means to drive the expression of the CRISPR Act3.0 dCas9 enzyme as a means to enhance target gene expression specifically in phloem tissues. To assess this approach more rapidly we developed a series of Arabidopsis transgenic lines with marker genes driven by phloem promoters of varying strength. Current studies are using these marker genes to both assess the ability of CRISPR-Act3.0 to enhance gene expression in a phloem specific manner as well as examine the optimal promoter regions for targeting the Act3.0 gRNAs. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. qPCR analysis of promoter edited protoplasts and calli for regenerated germplasm.As described above a protoplast transformation / gene editing process using the CRISPR-Cas12a ribonucleoprotein system was shown to be nearly 90% effective in editing a target gene sequence in citrus protoplasts. Additional studies have identified unique defense and phloem specific gene promoter sequences for use in subsequent multiplex CRISPR Act3.0 activation. Efforts in this task are ongoing and are focused on further investigating the efficacy of the Cas12a RNP system as well as establishing the Act3.0 gene activation system in our protoplast system. Task 2. Regeneration andin situexpression analysis of promoter edited calli and germplasm.Reliably embryogenesis and embryo germination protocols for citrus embryogenic lines have been successfully established. Globular embryos were derived from embryogenic callus after three to four weeks on solid MT-O media at 25°C in light. Once globular embryos formed, individual embryo could be transferred onto embryo germination media for development to the heart-shaped embryo stage and finally development to plantlets. To promote the formation of roots, plantlets were then transferred onto root induction media or micro-grafted onto Carrizo citrange rootstock. We also note a key bottleneck in this project is the time and efficiency associated with regenerating transformed plants. To address this concern, we are currently investigating the use of a newly developed CRISPR system, termed CRISPR-Combo, thatcan direct both targeted gene editing and the activation of morphogenic genes that enhance plant regeneration.In rice, CRISPR-Combo transgene directed morphogenic gene activation was shown to result in the regeneration of seedlings in hormone-free culture medium, with 83% of the regenerated plants carrying the targeted gene edit. No rice plants were regenerated in non-CRISPR-Combo controls. We are currently using aCarrizo citrange (Carrizo citrange trifoliate hybrid,Citrus sinensis"Washington" sweet orange XPoncirus trifoliate) transformation system to test the impact of activating a set of known plant morphogenic genes on citrus regeneration with the goal of using this information to enhance the efficiency and speed of our protoplast and embryogenic callus transformations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Shaun Curtin, Yiping Qi, L�zaro EP Peres, Alisdair R Fernie, Agustin Zs�g�n. Pathways to de novo domestication of crop wild relatives. Plant Physiology, 2021. https://doi.org/10.1093/plphys/kiab554
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Yingxiao Zhang, Yanhao Cheng, Hong Fang, Nathaniel Roberts, Liyang Zhang, Christopher A. Vakulskas, Randall P. Niedz, James N. Culver and Yiping Qi. 2022. Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases. Frontiers in Genome Editing, Vol.4 Article 780238, pp1-11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Yingxiao Zhang, Brian Iaffaldano, Yiping Qi. CRISPR ribonucleoprotein-mediated genetic engineering in plants. Plant Communications, 2021, 2 (2): 100168. https://doi.org/10.1016/j.xplc.2021.100168
- Type:
Book Chapters
Status:
Submitted
Year Published:
2022
Citation:
Hong Fang, James N. Culver, Randall P. Niedz, Yiping Qi. CRISPR/Cas21a-based Transgene-free Genome Editing in an Embryogenic Citrus Cell Line. Plant Genome Editing in Methods in Molecular Biology.
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:COVID19 restrictions, including hiring and space limitations slowed some experiments and has delayed bringing ona third post-doctoral student to work with Dr. Dardick, USDA-ARS, on gene and promoter analysis as well as asist with the production of gene editied citrus lines. Applicants for this position are currently being screened and the University returned to 100% occupancy this past June. These changes should rectify the notedlimitations. What opportunities for training and professional development has the project provided?Training opportunities in citrus tissue culture and gene analysis were providied for two postdoctoral students. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Efforts will continue as outlined under the objectives and timeline of the proposal. Specific ongoing and future efforts will focus on expanding protoplast gene editing efforts and demonstrating the regeneration of edited protoplasts into seedlings. Additional efforts will perform in planta characterizations of identified phloem and HLB induced promoters and promoter motifs as well as the use of the Act3.0 editing system to promote the expression of defense associated genes found to be induced in response to HLB.
Impacts
What was accomplished under these goals?
Objective 1. Development of multiplexed gene editing and gene activation methods in citrus.? Task 1. Establish citrus protoplast transformation systems. A stable callus maintenance procedure including both solid plate culture and liquid suspension culture has been developed.Citrus embryogenic callus lines (Citrus sinensis) under investigation in this study include: Hamlin 89 (H89), Hamlin 19 (H19), Valencia 17 (V17) and Pineapple 17 (Pa17). To make these cell lines more capable for protoplast isolation, citrus cell suspension culture were developed by crushing calli of each line in 50 mL liquid MT basal medium and incubating at 25°C in dark with 120 rpm shaking and a 21-day subculture cycle. Among these four cell lines, Hamlin cells (H89 and H19) developed much better than V17 and Pa17 lines. It is observed that, after 3-month's culture, Hamlin cells scatter evenly in suspension media while cells of Valencia and Pineapple lines stayed in clumps. Moreover, Hamlin lines, H89 line especially, display a high growth rate (doubling time about 4 to 5 days according to a rough estimationbased on fresh weight). These features make H89 a desired citrus cell line for protoplast isolation and further protoplast-based experiments. Citrus protoplast isolation and polyethylene glycol (PEG) mediated protoplast transfection has been established.Citrus cells from line H89 were used to produce protoplasts via standard isolation methods that include Cellulase R10 and Macerozyme R10 treatments that were capable of producing ~5×106live protoplasts per 0.8g of callus. A reporter construct carrying the GFP marker gene was subsequently used to test PEG mediated protoplast transfection. The average transfection efficiency was determined to be ~26%. Parameters such as protoplast numbers, target DNA concentration and incubation time of the transfection were optimized in order to promote greater transformation efficiency. Task 2. Develop multiplexed Cas12a system for promoter editing.? CRISPR-Cas12a ribonucleoprotein (RNP) mediatedtargeted mutagenesishas been established. A CRISPR-Cas12a based RNP delivery system has been tested on the protoplasts derived from citrus cells of line H89. The wild type Cas12a enzyme (LbCas12a, IDT Inc. USA) and the crRNA scaffolds based on the CRISPR-LbCpf system and targeting the citrus gene (CsPH5, GenBank ID 102616096) were pre-assembled with a molar ratio of 1:2 to form the RNP complex. The targeted gene edit is designed to disrupt a unique restriction enzyme site as a marker for identification.TheRNP complex was delivered to citrus protoplast through PEG mediated protoplast transfection which is the sameasthe transfection of reporter GFP construct described above.Afterthe transfection, protoplasts were incubated in the dark for 48 hr followed by restriction fragment length polymorphism (RFLP) assay. Different concentrations (0.1, 0.01 and 0.001 μM) of RNP complex were tested in the targeted mutagenesis of citrus protoplast. The result shows that the target site has been successfully mutated by the CRISPR-Cas12a based RNP delivery system and a 0.1 μM of RNP complex in the delivery system performs nearly 90% editing efficiency, which indicates the CRISPR-Cas12a based RNP delivery system is a powerful tool for citrus genome editing. Task 3. Develop CRISPR-Act3.0 for tissue-specific activation in phloem. CRISPR-Act3.0 enhancer constructs targeting the promoters of defense associated genes identified in objective 2 are currently being developed. Objective 2. Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming.? Task 1.In silicoidentification of target gene promoter sequences. Target defense genes and promoter motifs identified for gene editing.Meta-analysis of previous transcriptome studies on HLB sensitive, tolerant and resistant citrus cultivars were screened to identify gene transcripts that are significantly altered in response to HLB. This analysis has identified a set of defense associated genes that are significantly upregulated in resistant and tolerant cultivars but not in sensitive cultivars. We are currently working to increase the expression levels of these candidate genes in the phloem of HLB sensitive cultivars using the CRISPR Act 3.0 activation system. In addition, to identify potential promoter motifs needed to drive defense gene expression within the plants vascular phloem tissue, the site of HLB infection,we have first identified phloem specific genes via a meta-analysis of previous studies. Similar analysis were also performed on non-phloem specific genes, as a negative control, to confirm the specificity of phloem identified motifs. We have identified two motifs that show conservation within the promoters of putative phloem expressed citrus genes. These motifs will be examined for their ability to confer phloem expression in citrus. Task 2. Expression analysis and validation of identified target genes.? The above identified promoter motifs for both defense and phloem expression are currently being moved into expression vectors to test their ability to selectively drive the transcription of marker genes in citrus. In addition, these motifs will also be stacked to create a synthetic promoter to drive transcription of marker and defense genes in phloem, particularly in response to pathogen invasion. Objective 3. Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. Task 1. qPCR analysis of promoter edited protoplasts and calli for regenerated germplasm. As described above a protoplast transformation / gene editing process using the CRISPR-Cas12a ribonucleoprotein system was shown to be nearly 90% effective in editing a target gene sequence in citrus protoplasts.Additional studies have identified unique defense and phloem specific gene promoter sequences for multiplex subsequent CRISPR Act3.0 activation. Efforts in this task are ongoing and will focus on further investigating the efficacy of the Cas12a RNP system as well as establishing the Act3.0 gene activation system in our protoplast system. Task 2. Regeneration andin situexpression analysis of promoter edited calli and germplasm. Reliably embryogenesis and embryo germination protocols for citrus embryogenic lines have been successfully established.Globular embryos were derived from embryogenic callus after three to four weeks' incubation on solid MT-O media (MT basal media without any growth regulator) at 25°C in light. Once globular embryos formed, individual embryo could be transferred onto embryo germination media for development to the heart-shaped embryo stage and finally development to plantlets. To promote the formation of roots, plantlets were then transferred onto root induction media. After 2 to 3 weeks of root induction, plantlets with well-exhibited roots were able to be transferred to additional tissue culture media and then to soil.
Publications
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