Source: UNIV OF MARYLAND submitted to NRP
ENABLING EFFICIENT TRANSGENE-FREE PRECISE GENE EDITING IN CARROT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025828
Grant No.
2021-67013-34554
Cumulative Award Amt.
$290,000.00
Proposal No.
2020-05670
Multistate No.
(N/A)
Project Start Date
May 1, 2021
Project End Date
Apr 30, 2025
Grant Year
2021
Program Code
[A1191]- Agricultural Innovation through Gene Editing
Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Plant Science & Landscape Arch
Non Technical Summary
Carrot (Daucus carota) is a root vegetable often claimed to be the perfect health food. It is tasty and highly nutritious, containing high levels of β-carotene, fiber, potassium, antioxidants, vitamin K1 and vitamin B6. Carrots are found in different colors, such as yellow, white, orange, red and purple. Carrots are among the top 10 vegetables, based on global production records of primary vegetables based on FAO 2017 (http://www.fao.org/statistics). Interestingly, carrot production per capita in the Americas is only a third and a quarter to that of Oceania and Europe, respectively. It suggests ample room for production improvement as Americans are projected to eat a healthier diet, rich in nutritious vegetables. Carrot is a relatively recently domesticated vegetable crop. Recent sequencing of a carrot reference genome has paved the way for genetic improvements of carrot germplasms. As a promising breeding tool, gene editing is poised to revolutionize carrot breeding. Consequently, the development of robust carrot genome editing platforms has become urgent. Hence, it is important to develop a new carrot genome editing platform that is transgene-free. It will not only mitigate the regulatory burden, but also make it more appealing to consumers as we anticipate that genome editing will help accelerate the development of carrot germplasms that benefit consumers. While the genome editing technology is promised to accelerate crop breeding, its demonstration and optimization are lagging in breeding programs in the public sector. This is especially true for agriculturally important minor crops such as carrot. In this project, a transgene-free genome editing platform will be established in carrot. By applying highly efficient CRISPR-Cas12a tools to carrot through a transgene-free RNP delivery method, two complementary genome editing systems will be tested and established in carrots, one for high-efficiency targeted mutagenesis and the other for precise gene replacement. Given the broad applicability of the genome editing systems to be developed, the technologies to be developed in this project are potentially transformative as they can be applied to many other minor crops. This project specifically addresses the following two criteria of the program: 1) Transformation methods that do not require the use of plant pest sequences in plants or the development of gene editing technologies that do not require transformation; 2) If transformation technologies need to be developed, they must be coupled with the development of gene editing technologies. By demonstrating these new transgene-free genomes editing platforms and sharing the tools and protocols widely to other researchers and breeders, the success of this project will encourage adoption of these breeding technologies in public breeding sector, so that the goal set by USDA to increase US agriculture productivity by 40% while cutting the environmental footprint in half by 2050 can be met.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011452108050%
2011452108150%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1452 - Carrot;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
Objective 1: Develop efficient RNP delivery systems for targeted mutagenesis in carrotTo achieve efficient targeted mutagenesis in carrot, optimized Cas12a RNP delivery systems will first be established in carrot protoplasts. Three Cas12a of high editing performance will be used, including LbCas12a, LbCas12a-Ultra and AsCas12a-Ultra. Four genes will be chosen as the targets: 1) The phytoene desaturase gene (DcPDS) is a rate-limiting enzyme in carotenoid synthesis. Knockout of DcPDS will result in a photo-bleached phenotype that allows us to visualize targeted mutagenesis in carrot; 2) The lycopene epsilon-cyclase gene (DcLCYE) catalyzes the conversion from lycopene to α-carotene. Knockout of DcLCYE will shift lycopene metabolism toward the synthesis of β-carotene, provitamin A; 3) Dau c 1.01 and Dau c 1.02 are two genes encoding pathogenesis-related protein-10 (PR10) isoforms causing allergic reactions of carrots. Simultaneous knockout of these two genes can potentially create hypoallergenic carrots. Two crRNAs will be designed for each target gene using CRISPR-DT. The crRNAs will be preferred to locate near the 5'-terminus of coding sequences to have a higher chance to disrupt gene functions. The crRNAs with high predicted editing efficiencies and low potential off-targets will be used. All crRNAs will be synthesized as single-stranded RNAs with end protection and used for RNP delivery. Our first approach to obtain stably edited carrot plants is to regenerate from edited protoplasts. Our second approach to obtain stably edited carrot plants is to deliver Cas12a RNP into carrot calli. In both approaches, the crRNAs with the highest editing efficiencies for each target gene, along with the best performing Cas12a, will be used for RNP transformation. The Cas12a/crRNAs for Dau c 1.01 and Dau c 1.02 will be simultaneously delivered into protoplasts for a double knockout. For DcPDS or DcLCYE target gene, the best crRNA will be chosen for single target editing. The transformed protoplasts or calli will be maintained for plant regeneration, followed by genotyping of targeted mutants in the T0 generation.Objective 2: Develop an efficient HDR system with delivery of Cas12a/crRNA RNP and oligo donorsTo establish a transgene-free Cas12a-mediated HDR method in carrot, we will first conduct a series of experiments in carrot protoplasts to optimize the system. As a proof of concept, we will insert a small epitope tag, a 6XHis tag, to the C-terminus of DcPDS. Since PDS is a rate-limiting enzyme in carotenoid synthesis, a polyhistidine tag fusion is useful for studying PDS regulation. For example, it will allow us to quantify the PDS level at any circumstance, such as under biotic or abiotic stresses, or when other genes are up or down regulated, thus help us understand the regulatory network of carotenoid biosynthesis. The crRNA will be designed to locate at the C-terminus of the coding sequence. The optimized RNP delivery system obtained in the first objective will be used here. Donor sequence will harbor the inserted 6XHis tag sequence, as well as synonymous mutations to disrupt the crRNA targeting sequence or the PAM sequence, so that once the donor is incorporated into the genome, it will not be cut by Cas12a again. Multiple factors including dosage of oligo donors, donor strand, length of donors, and donor end protection and modification will be assessed to identify an optimized condition for highest HDR efficiency based on RNP delivery of Cas12a. The donor dosage and format that lead to the highest HDR efficiency will be applied to generate stably edited carrot plants, using the similar approaches described in the first objective. Since lower editing efficiency is expected for HDR-mediated precise genome editing, the transformation scale will be increased. Transformed protoplasts will be used to regenerate carrot plants, followed by genotyping of HDR events in the T0 generation.
Project Methods
Objective 1. Develop efficient RNP delivery systems for targeted mutagenesis in carrotOrange carrot cultivar Koral will be used as our plant material. Hypocotyls from two-week-old, sterilized seedlings will be used to isolate protoplasts, following previously developed protocols. About one million cells will be used for each protoplast transformation. Three biological replicates will be included for each treatment. Cas12a and crRNAs will be preassembled in vitro and delivered into protoplast using PEG-mediated transformation method. The optimized amount of RNPs and the Cas12a:crRNA ratio based on our data in rice will be applicated in carrot. After a two-day incubation in the dark, DNA will be isolated from carrot protoplasts and the targeted regions will be amplified by PCR. Amplicons will be submitted for next generation sequencing and the editing efficiency for each target will be calculated. Editing efficiencies from three versions of Cas12a will be compared and the best performing Cas12a will be further used to generate stably transformed plants.For the first approach based on protoplast transformation, RNP-transformed protoplasts will be transferred into the culture medium and embedded in a thin calcium alginate layer. The alginate layers with embedded protoplasts will be further transferred to the liquid culture medium (CPP medium) to culture for 3-6 weeks. After the formation of visible macrocolonies and somatic embryos, they will be released from alginate layers and placed on the solidified R medium for plant regeneration. DNA will be extracted from leaf tissues of regenerated carrots and used for target sites amplification. Editing results will be accessed using Sanger sequencing. Editing efficiencies will be calculated using the number of edited plants divided by regenerated plants. Edited plants will then be acclimatized and transferred to soil for phenotypic analysis. Plants with DcPDS knocked out should be easily identified by their photo-bleached phenotypes. Plants with DcLCYE knocked out will be used for carotenoid extraction. Lutein, α-carotene and β-carotene will be extracted from carrot roots and quantified by high performance liquid chromatography (HPLC) or by liquid chromatography-mass spectrometry (LC-MS). Plants with both Dau c 1.01 and Dau c 1.02 knocked out will be used for immunoblotting to detect the content of the allergenic protein PR-10. Wild type plants will be used as the control.For the second approach based on transformation of calli, carrot seeds will be sterilized and planted in MS medium. Hypocotyls will be excised as the explants and used to induce callus. The calli can be subcultured many times and maintained for RNP transformation. 10 µg of the best performing Cas12a, as well as the equal molar amount of the best performing crRNA for each target gene will be used to assemble RNPs. 0.75 mg gold particles will be coated with RNP and loaded onto two macrocarrier discs for particle bombardment. Non-coated gold particles will be used to generate control plants. For each RNP transformation, 50 calli will be bombarded based on previously published protocols. After transformation, calli will rest for two days and be transferred to MS medium for regeneration. Regenerated plantlets will be used for genome editing analysis and then moved to soil for phenotypic analysis.Objective 2: Develop an efficient HDR system with delivery of Cas12a/crRNA RNP and oligo donorsTo optimize the HDR method based on RNP delivery of Cas12a, the following four factors will be tested in this study to optimize donor delivery: 1) Dosage of oligo donors. Three donor amounts, 0.5 nmol, 1 nmol and 2 nmol, will be co-delivered into carrot protoplasts together with Cas12a/crRNA RNPs. 2) Donor strand. HDR donors will be delivered as ssODN with the sequence of the template strand or the non-template strand. As crRNA binds to the non-template strand of genes, donor strand can have a significant effect on repair efficiency by HDR. The double-stranded DNA (dsDNA) will not be included as the repair donors, since dsDNA has a higher potential to insert into genome randomly, causing unexpected transgene events. 3) Length of donors. Three donor lengths, 50 nt, 100 nt and 200 nt, will be tested. A longer donor may increase the editing efficiency but is difficult to synthesize and deliver. 4) Donor end protection and modification. Chemically modified ssODN can increase donor stability, thus increase editing efficiency. Donors without any modification and with end protection and modification will be tested here for comparison. Transformed protoplast will be incubated in the dark for two days and used for DNA extraction, PCR amplification and deep sequencing as described in the first objective.The methods established in Objective 1 for carrot plant regeneration will be used for generating HDR plants based on RNP delivery of Cas12a and donors. DNA from 10 regenerated calli will be pooled for detection of HDR events by PCR and only positive pools will be carried to full plant regeneration. Later, DNA will be extracted from individual regenerated plants for PCR and Sanger sequencing to further screen for HDR events. Western blot will be used to detect PDS-6XHis with an anti-his tag antibody in HDR-positive plants.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity in carrot will directly benefit carrot breeding and shed light on applying genome editing to improve other vegetable crops. The efficient CRISPR-Cas12a-based targeted mutagenesis and gene replacement systems will provide the breeders in the public sector and private industry useful technologies for achieving their product development goals. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations: 1. March 7, 2024. aBiotech Journal Webinar. 2. March 5, 2024. Recombination and Genome Editing in Plants. Federation of American Societies for Experimental Biology (FASEB). 3. February 22, 2024. 6th CRISPR AgBio Congress. Theme: Engineer the Next Generation of Agriculture. 4. February 20, 2024. Enhancing the Global Food System's Resilience to Biological Threats. Scowcroft Institute of International Affairs, The BUSH School, TAMU. 5. January 30, 2024, Maximizing Agriculture to Enhance Nutrient Composition to Better Fulfill Dietary Recommendations. Workshop by National Academies of Sciences, Engineering, and Medicine. 6. January 14, 2024, Plant and Animal Genome Conference. Organellar Genome Engineering Workshop. San Diego, CA. 7. January 14, 2024, Plant and Animal Genome Conference. Development and Application of Genome Engineering and Transgenic Technology to the Agriculture Workshop. San Diego, CA. 8. October 13, 2023. Department of Plant Biology, Rutgers University, NJ. 9. September 29, 2023. School of Life Sciences, University of Nevada, Las Vegas, NV. 10. July 13, 2023. Gates Masterclass on construct design for crop engineering, gene regulation and genome editing. Online. The audience of these presentations was very diverse, including students, academic researchers, industry researchers and governmental officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This projecthas provided training opportunities for one postdoc (Yanhao Cheng), one visiting PhD student (Nayara Sabrina de Freitas Alves), and two high school students (Christina Zhang from Eleanor Roosevelt High School and Doris Wang from Montgomery Blair High School). How have the results been disseminated to communities of interest?We have developed highly efficient LbCas12a for genome editing in plants, either through plasmid delivery or RNP delivery. These important reagents were recently reported in the journal Genome Biology (2023) 24:102 and the plasmids have been made available to the research community through Addgene (https://www.addgene.org/Yiping_Qi/). What do you plan to do during the next reporting period to accomplish the goals?As we have identified the most potent LbCas12a-RRV variant, selected the appropriate carrot varieties, and optimized the protoplast transformation condition, we will use this variant (in comparison to the wildtype LbCas12a) to achieve NHEJ-based mutagenesis and HDR- or NHEJ- based gene replacement in carrot cells in the remainder of the project.

Impacts
What was accomplished under these goals? Carrot is an agriculturally important minor crop and is among the top 10 highly consumed vegetables in the world. The carrot genome has been recently sequenced which opens the door for genome editing based breeding. However, technological barriers exist which impede the use of gene-editing in carrot. In this project, we propose to develop transgene-free genome editing systems in carrot based on ribonucleoprotein (RNP) delivery of Cas12a protein and crRNA. Unlike most CRISPR-Cas delivery systems, RNP delivery does not rely on transgene and hence is regulation-friendly. Due to its transient nature, RNP delivery can also mitigate potential off-target effects. The primary goal of this proposal is to develop transgene-free genome editing systems for precise gene knockout and gene replacement in carrot, enabling basic and applied research in this important and versatile vegetable crop. In Objective 1, we will compare multiple promising Cas12a orthologs to develop an efficient targeted mutagenesis system in carrot. In Objective 2, we will develop a precise gene replacement system in carrot. This project aligns well with the goals of the "Agricultural Innovation through Gene Editing" program (Area Priority Code: A1191). The innovative gene editing systems that we aim to establish will also enable similar applications in other agriculturally important minor crops. The success of this project will encourage adoption of these breeding technologies in public breeding sector, so that the goal set by USDA to increase US agriculture productivity by 40% while cutting the environmental footprint in half by 2050 can be met. Objective 1. Develop efficient RNP delivery systems for targeted mutagenesis in carrot (1) Major activities completed: A few carrot varieties (B493, Imperator 58, Atomic red, Baltimore Hybrid, Royal Chantenay, Scarlet Nantes, and Yaya Hybrid) were used for an initial assessment of protoplast isolation and transformation with a GFP reporter vector. The highest protoplast transformation efficiency was around 20%. Meanwhile, five engineered LbCas12a variants (LbCas12a-D156R, LbCas12a-RV, LbCas12a-RRV, LbCas12a-RVQ, and LbCas12a-RRVQ) were compared to the wildtype LbCas12a nuclease in rice and tomato protoplasts to identify the best performing Cas12a nuclease for use in carrot cells. The last two LbCas12a variants came from a research collaboration between PD and IDT. Specifically, LbCas12a, LbCas12a-D156R, and LbCas12a-RVQ were compared using RNP delivery. The performance of these Cas12a nucleases in different temperatures (25ºC and 32ºC) was also assessed. (2) Data collected: Initial data on protoplast yield and transformation efficiency using different tissues (e.g., etiolated seedlings and cotyledons) was assessed. Data for genome editing efficiencies and targeted mutagenesis profiles in rice and tomato genomes by transforming protoplasts with either RNP or plasmids using different reagent dosages and targeting multiple sites with different PAM (protospacer adjacent motif) requirements under temperature conditions of 25ºC and 32ºC were also evaluated. The genome editing frequencies were measured using next-generation sequencing (NGS) of PCR amplicons. Concomitantly, genome editing efficiency in stably transformed plants (rice and poplar) was collected. (3) Summary statistics and discussion of results: Three LbCas12a nucleases were compared targeting two TTTV (V=A,C,G) PAM sites and two TTV sites in rice protoplasts at 0.001 uM RNP and 0.01 uM RNP concentrations. The data showed that LbCas12a-D156R and LbCas12a-RVQ generated up to 50% editing frequency at the TTTV PAM sites and up to 8% editing frequency at the TTV PAM sites. These three LbCas12a nuclease were also compared in tomato protoplasts at six independent TTTV PAM sites. High genome editing efficiency was observed for LbCas12a-RVQ, with ~60% editing frequency for six target sites, followed by LbCas12a-D156R, with xx% editing efficiency for x sites. Hence, LbCas2a-D156R and LbCas12a-RVQ were chosen for ongoing RNP delivery research for this Objective as well as for Objective 2. (4) We further conducted experiments to assess the efficacy of CRISPR-Cas9-mediated plasmid delivery in carrot protoplasts. Next-generation sequencing (NGS) was employed to detect genome editing, which revealed an efficiency rate of approximately 3%-4% across various genomic sites. Therefore, it is anticipated that utilizing RNP delivery could significantly enhance this efficiency. Our future investigations will delve into RNP-mediated delivery mechanisms to further optimize the gene-editing protocol in carrot protoplasts to achieve more robust and efficient genome modification outcomes. (5) Our research team has also successfully developed a protocol for inducing callus formation and achieving stable transformation in carrot tissue. This process utilizes 'Ruby' and GFP reporter genes, which enables the visual identification of successful transformations by the purple coloration and green fluorescence (digital Axiocam) in regenerated calli, respectively. Furthermore, we are currently in the process of establishing and refining a system for plant regeneration from carrot protoplasts. Objective 2: Develop an efficient HDR system with the delivery of Cas12a/crRNA RNP and oligo donors (1) Major activities completed: crRNA have been designed and synthesized for phytoene desaturase gene (DcPDS, DCAR_016085), lycopene epsilon-cyclase gene (DcLCYE, DCAR_028276), and Dau c 1.01 and Dau c 1.02 that encode pathogenesis-related protein-10 (PR10) isoforms causing allergic reactions to carrots. We designed and synthesized double-stranded oligodeoxynucleotide donors (dsODNs) harboring stop code with 5' and 3' phosphorothioate modifications. Multiple carrot varieties have been tested to compare transformation efficiency in protoplasts. (2) Data collected: Transformation efficiencies in the protoplasts of a few carrot varieties. (3) Summary statistics and discussion of results: We further improved protoplast transformation efficiency to ~50% for carrot varieties B493 and imperator 58. (4) Key outcomes or other accomplishments realized: We published a Method in Mol Biol book chapter on RNP delivery of CRISPR-Cas12a into protoplasts of dicot plants. We also identified an NHEJ-based alternative strategy to achieve gene insertion should our HDR approach fail. (5) We designed and synthesized the oligos with 5' phosphorylation and phosphorothioate modification, then paired oligos were annealed to produce dsODNs with sticky ends in different lengths. We prepared the dsODN donors of HA, 3xflag, and GFP. Owing to the length limitation of oligo synthesis, the GFP dsODN donors were prepared by PCR with the corresponding chemically modified primers. PCR products were purified and digested for further usage.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 3. Simon Sretenovic#, Yumi Green, Yuechao Wu, Yanhao Cheng#, Tao Zhang*, Joyce Van Eck*, Yiping Qi*. Genome-and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato. Plant Physiology. 06/14/2023. https://doi.org/10.1093/plphys/kiad347
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 9. Yingxiao Zhang #, Yuechao Wu, Gen Li #,, Aileen Qi##,, Yong Zhang, Tao Zhang*, Yiping Qi*. Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice. The Plant Genome. 2023. https://doi.org/10.1002/tpg2.20266
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 1. Aytug Tuncel#, Changtian Pan, Thorben Sprink, Ralf Wilhelm, Rodolphe Barrangou, Li Li, Patrick M Shih, Rajeev K Varshney, Leena Tripathi, Joyce Van Eck, Kranthi Mandadi, Yiping Qi*. Genome-edited foods. Nature Reviews Bioengineering. Published: 04 October 2023. https://www.nature.com/articles/s44222-023-00115-8
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 4. Gen Li#, Yingxiao Zhang#, Micah Dailey#, Yiping Qi*. Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants. Frontiers in Genome Editing. 2023 Oct 12:5:1251903. https://www.frontiersin.org/articles/10.3389/fgeed.2023.1251903/full
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 2. Zhaohui Zhong, Guanqing Liu, Zhongjie Tang, Shuyue Xiang, Liang Yang, Lan Huang, Yao He, Tingting Fan, Shishi Liu, Xuelian Zheng, Tao Zhang, Yiping Qi*, Jian Huang*, Yong Zhang*. Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system. Nature Communications. 2023 Sep 29;14(1):6102. https://www.nature.com/articles/s41467-023-41802-9


Progress 05/01/22 to 04/30/23

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity in carrot will directly benefit carrot breeding and shed light on applying genome editing to improve other vegetable crops. The efficient CRISPR-Cas12a based targeted mutagenesis and gene replacement systems will provide the breeders in the public sector and private industry useful technologies for achieving their product development goals. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations which mostly occurred virtually due to the Covid-19 pandemic: 1. March 17, 2023. Department of Genetics and Biochemistry. Clemson University, Clemson, SC. 2. February 14,2023. The 5th Annual CRISPR AgBio Congress, San Diego, CA. 3. February 7, 2023. Center for Computational and Integrative Biology, Rutgers-Camden, New Jersey. 4. January 19, 2023. International Seminar and Workshop on CRISPR/Cas-based Plant Functional Genomics and Computational Modeling, Jan 18-21, 2023. Jorhat, Assam, India. 5. January 15, 2023. Plant and Animal Genome Conference, Jan 13-18, 2023. San Diego, CA. 6. November 18, 2022. The 2nd International Symposium for Horticultural Plant Biology and Biotechnology. Beijing, China. 7. October 31, 2022. The 5th International Conference on CRISPR Technologies. Berkeley, CA. 8. October 24, 2022. The 9th Plant Genomics and Gene Editing Congress (USA). Raleigh, NC. 9. September 14, 2022. The Center of Soybean Research of the Chinese University of Hong Kong and USDA's Agricultural Trade Office (ATO) in Hong Kong. 10. August 30, 2022. Rubber Research Institute, Chinese Academy of Tropical Agricultural Science. 11. August 10, 2022. The 3rd International Symposium of Horticulture and Plant Biology, Huazhong Agriculture University, Wuhan. 12. July 29, 2022. 2022 International Symposium on Plant Biotic Interactions and Plant Health, Wuhan, China. 13. July 10, 2022. Plant Biology 2022. ASPB and CSPB/SCBV. Portland, USA. 14. July 7, 2022. The 20th IUFRO Tree Biotech Meeting, Harbin, China. 15. June 29, 2022. PlantEd COST Action - plant genome editing. European Cooperation in Science and Technology. 16. June 7, 2022. 2022 In Vitro Biology Meeting, San Diego, CA. 17. June 5, 2022. 2022 In Vitro Biology Meeting, San Diego, CA. 18. May 29, 2022. The 23rd Penn State Symposium in Plant Biology -- RNA Biology. State College, PA. 19. May 25, 2022. Novel Crop Breeding Techniques: Towards more sustainable agriculture (http://plantepigenetics.ch/NBT2022/), ERC Consolidator Project BUNGEE 725701. The audience of these presentations was very diverse, including students, academic researchers, industry researchers and governmental officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project so far has provided training opportunities for one postdoc, Yanhao Cheng, in the Qi lab. How have the results been disseminated to communities of interest?We have developed highly efficient LbCas12a for genome editing in plants, either through plasmid delivery or RNP delivery. These important reagents were recently reported Genome Biology (2023) 24:102 and the plasmids have been made available to the research community through Addgene (https://www.addgene.org/Yiping_Qi/). What do you plan to do during the next reporting period to accomplish the goals?As we have identified the most potent LbCas12a-RRV variant, selected the appropriate carrot varieties, and optimized the protoplast transformation condition, we will use this variant (in comparison to the wildtype LbCas12a) to achieve NHEJ-based mutagenesis and HDR or NHEJ-based gene replacement in carrot cells in the remainder of the project.

Impacts
What was accomplished under these goals? Carrot is an agriculturally important minor crop and is among the top 10 highly consumed vegetables in the world. The carrot genome has been recently sequenced which opens the door for genome editing based breeding. However, technological barriers exist which impede the use of gene-editing in carrot. In this project, we propose to develop transgene-free genome editing systems in carrot based on ribonucleoprotein (RNP) delivery of Cas12a protein and crRNA. Unlike most CRISPR-Cas delivery systems, RNP delivery does not rely on transgene and hence is regulation-friendly. Due to its transient nature, RNP delivery can also mitigate potential off-target effects. The primary goal of this proposal is to develop transgene-free genome editing systems for precise gene knockout and gene replacement in carrot, enabling basic and applied research in this important and versatile vegetable crop. In Objective 1, we will compare multiple promising Cas12a orthologs to develop an efficient targeted mutagenesis system in carrot. In Objective 2, we will develop a precise gene replacement system in carrot. This project aligns well with the goals of the "Agricultural Innovation through Gene Editing" program (Area Priority Code: A1191). The innovative gene editing systems that we aim to establish will also enable similar applications in other agriculturally important minor crops. The success of this project will encourage adoption of these breeding technologies in public breeding sector, so that the goal set by USDA to increase US agriculture productivity by 40% while cutting the environmental footprint in half by 2050 can be met. Objective 1. Develop efficient RNP delivery systems for targeted mutagenesis in carrot (1) Major activates completed: A few carrot varieties (B493, Imperator 58, Atomic red, Baltimore Hybrid, Royal Chantenay, Scarlet Nantes and Yaya Hybrid) were used for the initial assessment of protoplast generation and transformation with a GFP reporter. Highest protoplast transformation efficiency was close to 20%. Meanwhile, three engineered LbCas12a variants (LbCas12a-D156R, LbCas12a-RVQ, LbCas12a-RV, and LbCas12a-RRV) were compared to the wildtype LbCas12a nuclease in rice and tomato protoplasts, to identify the best performing Cas12a to be used for carrot cells. The latter two LbCas12a variants were resulted from a research collaboration between the PD and IDT. Specifically, LbCas12a, LbCas12a-RV, and LbCas12a-RVQ were compared using RNP delivery. Performance of these Cas12a nucleases at different temperatures (25ºC and 32ºC) was also assessed. (2) Data collected: Initial data of protoplast yield using different tissues (e.g., etiolated seedlings and cotyledons) and transformation efficiency were collected. Data for genome editing efficiencies and targeted mutagenesis profiles in rice and tomato genomes by transforming protoplasts with either RNP or plasmids at different reagent dosages and at multiple target sites with different PAM (protospacer adjacent motif) requirements at 25ºC and 32ºC were also collected. The genome editing frequencies were measured using next generation sequencing (NGS) of PCR amplicons. Genome editing efficiency in stably transformed plants (rice and poplar) has also been collected. (3) Summary statistics and discussion of results: Three LbCas12a nucleases were compared at targeting two TTTV (V=A,C,G) PAM sites and two TTV PAM sites in rice protoplasts at 0.001 uM RNP and 0.01 uM RNP concentrations. The data showed that LbCas12a-D156R and LbCas12a-RV generated up to 50% editing frequency at the TTTV PAM sites and up to 8% editing frequency at the TTV PAM sites. These three LbCas12a nuclease were also compared in tomato protoplasts at six independent TTTV PAM sites. Very high genome editing efficiency was observed for LbCas12a-RV, with ~60% editing frequency for six target sites, followed by LbCas12a-D156R. LbCas12a-RRV showed the highest genome editing efficiency (up to 100%) in both rice and poplar. (4) Key outcomes or other accomplishments realized: It is discovered that LbCas12a-RVQ showed the most robust genome editing efficiency that the previously reported temperature-tolerant (tt) LbCas12a-D156R variant. LbCas12a-RRV resulted in 1.5 to 10-fold improvement on genome editing efficiency over the wildtype LbCas12a based on assessment in protoplasts of rice and tomato, and this improvement is expected to be transferrable into carrot protoplasts. These results suggest that LbCas12a-RRV, not the LbCas12a-RVQ identified earlier, represents a most efficient Cas12a nuclease for plant genome editing. Hence, this Cas12a-RRV is chosen for ongoing research in this Objective as well as Objective 2. Objective 2: Develop an efficient HDR system with the delivery of Cas12a/crRNA RNP and oligo donors (1) Major activities completed: crRNA have been designed and synthesized for phytoene desaturase gene (DcPDS, DCAR_016085), lycopene epsilon-cyclase gene (DcLCYE, DCAR_028276), and Dau c 1.01 and Dau c 1.02 that encode pathogenesis-related protein-10 (PR10) isoforms causing allergic reactions of carrots. We designed and synthesized double-stranded oligodeoxynucleotide donors (dsODNs) harboring stop code with 5' and 3' phosphorothioate modifications. Multiple carrot varieties have been further tested to compare transformation efficiency in protoplasts. (2) Data collected: Transformation efficiencies in the protoplasts of a few carrot varieties. (3) Summary statistics and discussion of results: We further improved protoplast transformation efficiency to ~50% for carrot varieties B493 and imperator 58. (4) Key outcomes or other accomplishments realized: We published a Methods in Mol Biol book chapter on RNP delivery of CRISPR-Cas12a into protoplasts of a dicot plants. We also identified an NHEJ-based alternative strategy to achieve gene insertion should our HDR approach fail.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan#, Gen Li#, Aimee A. Malzahn#, Benjamin Leyson##, Simon Sretenovic#, Yanhao Cheng#, Filiz Gurel#, Gary D. Coleman, Yiping Qi*. Boosting plant genome editing with a versatile CRISPR-Combo system. Nature Plants, 2022. 8(5):513-525. https://www.nature.com/articles/s41477-022-01151-9
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan#, Yiping Qi*. A CRISPR-Combo method for improving genome editing in plants. Nature Protocols. 2023. https://www.nature.com/articles/s41596-023-00823-w
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Yanhao Cheng#, Yingxiao Zhang#, Gen Li#, Hong Fang#, Simon Sretenovic#, Avery Fan#, Jiang Li, Jianping Xu, Qiudeng Que, Yiping Qi*. CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice. Plant Communications. 2023. https://doi.org/10.1016/j.xplc.2023.100601
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Changtian Pan#, Gen Li#, Anindya Bandyopadhyay, Yiping Qi*. Guide RNA library based CRISPR screens in plants: opportunities and challenges. Current Opinion in Biotechnology, 2023, 79: 102883
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Aytug Tuncel*, Yiping Qi*. CRISPR/Cas mediated genome editing in potato: Past achievements and future directions. Plant Science. 2022. https://doi.org/10.1016/j.plantsci.2022.111474
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Shishi Liu, Simon Sretenovic, Tingting Fan, Yanhao Cheng, Gen Li, Aileen Qi, Xu Tang, Yang Xu, Weijun Guo, Zhaohui Zhong, Yao He, Yanling Liang, Qinqin Han, Xuelian Zheng, Xiaofeng Gu, Yiping Qi*, Yong Zhang*. Hypercompact CRISPR-Cas12j2 (Cas?) enables genome editing, gene activation, and epigenome editing in plants. Plant Communications. 2022. https://doi.org/10.1016/j.xplc.2022.100453
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Naga Rajitha Kavuri, Manikandan Ramasamy, Yiping Qi, Kranthi Mandadi. Applications of CRISPR/Cas13-Based RNA Editing in Plants. Cells. 2022. 11 (17), 2665. https://www.mdpi.com/2073-4409/11/17/2665
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Liyang Zhang, Gen Li#, Yingxiao Zhang#, Yanhao Cheng#, Steve Glenn, Michael Collingwood, Nicole Bode, Sarah Beaudoin, Christopher Vakulskas*, Yiping Qi*. Robust genome editing by engineered LbCas12a variants in human cells and plants. Genome Biology. 2023. 24, Article number: 102. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-023-02929-6
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ayman Eid#, Yiping Qi*. Prime editor integrase systems boost targeted DNA insertion and beyond. Trends in Biotechnology. 2022, 40 (8): 907-909. https://doi.org/10.1016/j.tibtech.2022.05.002


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:This project will provide cutting-edge training on plant genome editing technology development and evaluation for a post-doc, as well as multiple undergraduate and high school interns. The new CRISPR genome editing systems with improved editing activity in carrot will directly benefit carrot breeding and shed light on applying genome editing to improve other vegetable crops. The efficient CRISPR-Cas12a based targeted mutagenesis and gene replacement systems will provide the breeders in the public sector and private industry useful technologies for achieving their product development goals. In this reporting period, the PI has presented the work and acknowledged this NIFA funding in the following presentations which mostly occurred virtually due to the Covid-19 pandemic: 1. May 27-28, 2021, 2021 Joint MA-ASPB and UMD Plant Minisymposium; 2. June 6, 2021. SIVB 2021: In Vitro OnLine. Session: Variables Controlling Successful Gene Editing in Plants/New Tools; 3. July 21, 2021. Reliance Industries Limited (RIL) R&D Community Webinar Series.; 4. July 28, 2021. Genome Writers Guild (GWG) Virtual Conference. University of Minnesota; 5. August 17, 2021. CTC Genomics, St. Louis, Missouri; 6. September 2, 2021. National Center for Genome Editing in Agriculture, Israel; 7. September 14, 2021. Virtual Workshop on Advanced Biotechnology in Bosnia and Herzegovina; 8. September 15, 2021. 2nd Congress of Geneticists in Bosnia and Herzegovina with International Participation; 9. Oct 7-10, 2021. 12th International Scientific Agriculture Symposium "AGROSYM 2021", Bosnia and Herzegovina; 10. November 7-10, 2021 ASA, CSSA,SSSA International Annual Meeting, Salt lake City, UT; 11. January 10, 2022. Plant and Animal Genome XXIX Conference, Jan 8-12, 2022. San Diego, CA; 12. February 10, 2022. Dale Bumpers National Rice Research Center-USDA-ARS. Stuttgart, AR. The audience of these presentations was very diverse, including students, academic researchers, industry researchers and governmental officials from different countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project so far has provided training opportunities for two postdocs, Gen Li and Yanhao Cheng, in the Qi lab. 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?The research has been affected by the delayed hiring of a postdoc dedicated to this project. Due to the Covid-19 pandemic and international travel restrictions, it is very hard to identify and hire a suitable postdoc to take on this project. The postdoc Gen Li initiated some of the LbCas12a variants comparison work. Luckily, the newly graduated PhD student (Yanhao Cheng) in Qi lab recently agreed to stay in Qi lab and work on this project as a postdoc. She is a very experienced young scientist in genome editing and plant tissue culture. It is anticipated that steady progress will be made down the road toward the two Objectives.

Impacts
What was accomplished under these goals? Carrot is an agriculturally important minor crop and is among the top 10 highly consumed vegetables in the world. The carrot genome has been recently sequenced which opens the door for genome editing based breeding. However, technological barriers exist which impede the use of gene-editing in carrot. In this project, we propose to develop transgene-free genome editing systems in carrot based on ribonucleoprotein (RNP) delivery of Cas12a protein and crRNA. Unlike most CRISPR-Cas delivery systems, RNP delivery does not rely on transgene and hence is regulation-friendly. Due to its transient nature, RNP delivery can also mitigate potential off-target effects. The primary goal of this proposal is to develop transgene-free genome editing systems for precise gene knockout and gene replacement in carrot, enabling basic and applied research in this important and versatile vegetable crop. In Objective 1, we will compare multiple promising Cas12a orthologs to develop an efficient targeted mutagenesis system in carrot. In Objective 2, we will develop a precise gene replacement system in carrot. This project aligns well with the goals of the "Agricultural Innovation through Gene Editing" program (Area Priority Code: A1191). The innovative gene editing systems that we aim to establish will also enable similar applications in other agriculturally important minor crops. The success of this project will encourage adoption of these breeding technologies in public breeding sector, so that the goal set by USDA to increase US agriculture productivity by 40% while cutting the environmental footprint in half by 2050 can be met. Objective 1. Develop efficient RNP delivery systems for targeted mutagenesis in carrot (1) Major activates completed: A few carrot varieties (Imperator 58, Lunar White, Purple Dragon, Red, Solar Yellow, and Tendersweet) were used for the initial assessment of protoplast generation and transformation with a GFP reporter. The highest protoplast transformation efficiency was close to 20%. Meanwhile, three engineered LbCas12a variants (LbCas12a-D156R, LbCas12a-RVQ, and LbCas12a-RRVQ) were compared to the wildtype LbCas12a nuclease in rice and tomato protoplasts, to identify the best performing Cas12a to be used for carrot cells. The latter two LbCas12a variants resulted from a research collaboration between the PD and IDT. Specifically, LbCas12a, LbCas12a-D156R, and LbCas12a-RVQ were compared using RNP delivery. The performance of these Cas12a nucleases at different temperatures (25ºC and 32ºC) was also assessed. (2) Data collected: Initial data of protoplast yield using different tissues (e.g., etiolated seedlings and cotyledons) and transformation efficiency were collected. Data for genome editing efficiencies and targeted mutagenesis profiles in rice and tomato genomes by transforming protoplasts with either RNP or plasmids at different reagent dosages and at multiple target sites with different PAM (protospacer adjacent motif) requirements at 25ºC and 32ºC were also collected. The genome editing frequencies were measured using next-generation sequencing (NGS) of PCR amplicons. (3) Summary statistics and discussion of results: Three LbCas12a nucleases were compared at targeting two TTTV (V=A,C,G) PAM sites and two TTV PAM sites in rice protoplasts at 0.001 uM RNP and 0.01 uM RNP concentrations. The data showed that LbCas12a-D156R and LbCas12a-RVQ generated up to 50% editing frequency at the TTTV PAM sites and up to 8% editing frequency at the TTV PAM sites. These three LbCas12a nucleases were also compared in tomato protoplasts at six independent TTTV PAM sites. Very high genome editing efficiency was observed for LbCas12a-RVQ, with ~60% editing frequency for six target sites, followed by LbCas12a-D156R. (4) Key outcomes or other accomplishments realized: It is discovered that LbCas12a-RVQ showed the most robust genome editing efficiency that the previously reported temperature-tolerant (tt) LbCas12a-D156R variant. LbCas12a-RVQ resulted in a 1.5 to 10-fold improvement in genome editing efficiency over the wildtype LbCas12a based on assessment in protoplasts of rice and tomato, and this improvement is expected to be transferrable into carrot protoplasts. These results suggest that LbCas12a-RVQ probably represents the most efficient Cas12a nuclease for plant genome editing. Hence, this Cas12a-RVQ is chosen for ongoing research in this Objective as well as Objective 2. Objective 2: Develop an efficient HDR system with delivery of Cas12a/crRNA RNP and oligo donors We have not started working on this objective. Updates will be provided in the future.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 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: Md. Mahmudul Hassan, Yingxiao Zhang, Guoliang Yuan, Kuntal De, Jin-Gui Chen, Wellington Muchero, Gerald A. Tuskan, Yiping Qi, Xiaohan Yang. Construct design for CRISPR/Cas-based genome editing in plants. Trends in Plant Science, 2021, https://www.cell.com/trends/plant-science/fulltext/S1360-1385(21)00157-6
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Kutubuddin Molla, Simon Sretenovic, Kailash C Bansal, Yiping Qi. Precise plant genome editing with base editors and prime editors. Nature Plants, 2021, 7: 1166-1187. https://www.nature.com/articles/s41477-021-00991-1