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Projects / Programmes source: ARIS

Genome editing of selected Brassica species with CRISPR/Cas9

Research activity

Code Science Field Subfield
4.03.00  Biotechnical sciences  Plant production   

Code Science Field
B225  Biomedical sciences  Plant genetics 

Code Science Field
4.01  Agricultural and Veterinary Sciences  Agriculture, Forestry and Fisheries 
Keywords
CRISPR/Cas9, Brassica, genome editing, next-generation sequencing, disease resistance, CENH3, targeted mutagenesis, NHEJ, HDR
Evaluation (rules)
source: COBISS
Researchers (13)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  09565  PhD Borut Bohanec  Biotechnology  Retired researcher  2018 - 2022  592 
2.  50811  Tjaša Cesar  Plant production  Technical associate  2018 - 2020  13 
3.  53354  Petra Dekleva    Technical associate  2019 - 2020 
4.  17787  Viktorija Dolenc    Technical associate  2018 - 2019 
5.  31143  Nataša Hren    Technical associate  2018 - 2019 
6.  28881  PhD Karolina Ivičak Kocjan  Biotechnology  Researcher  2018 - 2020  54 
7.  06628  PhD Roman Jerala  Biochemistry and molecular biology  Researcher  2018 - 2022  1,189 
8.  24413  PhD Jana Murovec  Biotechnology  Head  2018 - 2022  169 
9.  53734  Peter Pečan  Biochemistry and molecular biology  Researcher  2020 - 2022  17 
10.  50616  Arne Praznik  Biochemistry and molecular biology  Researcher  2018 - 2022  35 
11.  52178  Miha Slapnik  Plant production  Researcher  2019 - 2022  19 
12.  38120  PhD Ester Stajič  Plant production  Junior researcher  2018 - 2020  42 
13.  54400  Sinja Svetik  Biotechnology  Technical associate  2020 - 2022 
Organisations (2)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,932 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,273 
Abstract
BACKGROUND The genus Brassica comprises a large number of species and subspecies that are consumed either as shoots, leaves, roots, turnip roots or in the form of seeds. Vegetative plant parts are merchandized mainly as raw products, whereas generative parts in a processed form either as oil, meal, powder, protein, condiment, etc. The species have diversified into a large number of agriculturally important morphotypes due to domestication and further breeding. Nowadays, the species B. oleracea comprises morphotypes of cabbage, kale, Chinese kale, savoy cabbage, Brussel sprout, kohlrabi, broccoli, cauliflower; the species B. rapa includes morphotypes of pak choi, Chinese cabbage, turnip, and oilseeds. The species B. napus, an allopolyploid, also includes several morphotypes (rapeseed, rutabaga, fodder rape), with rapeseed (canola) being the economically most important as the third oil crop regarding production quantity. Despite their high economic importance, modern biotechnological approaches for breeding and research of Brassica species are still lacking. Few published studies on genome editing relied on stable integration of genome editing vectors with A. tumefaciens, which limits their applicability in horticulture. Although the introduced DNA can be segregated out after mutagenesis, the use of transgenesis during plant variety development can still trigger GMO regulation in countries that rely on process-based regulatory approaches. Therefore, we believe that new protocols for DNA-integration-free genome editing of Brassica species are needed and that the use of preassembled sgRNA-Cas9 ribonucleoprotein complexes (RNPs) provides an optimal solution. RNPs are exceptional comparing to other expression technologies as they enable DNA-free genome editing. Moreover, the introduction of RNPs generates genome modifications faster as compared to plasmid delivery since they don’t need to be transcribed and translated in cells. They are also degraded faster than other vectors, thus limiting exposure to genome editing reagents. It results in fewer off-target modifications, which are currently the biggest drawback of the CRISPR/Cas9 technique. Delivery of RNPs also prevents insertional mutagenesis as no vector is integrated at random into the genome. AIM The main goal of the project is to develop genome editing approaches for Brassica species that will rely on delivery of RNPs into protoplasts and microspores and regeneration of edited plants. IMPACT The project will combine basic science with a final application in horticulture. The results of the proposed project will have an impact on the implementation of cutting-edge technology in the fields of plant breeding and plant biotechnology and will have a substantial impact on their further development, as we plan to be the first to: 1. Use Cas9-sgRNA ribonucleoprotein complexes (RNPs) for genome editing of any Brassica species. 2. Use RNPs for genome editing of haploid gametes (microspores) of any plant species. 3. Use RNPs in combination with donor DNA for induction of homology-dependent repair (HDR) in plants. 4. Introduce RNPs into plant cells (protoplasts and microspores) by two innovative approaches: with the use of cell penetrating peptides (CPPs) and electroporation. 5. Use CRISPR/Cas9 for development of cabbage plants resistant to Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson. 6. Use CRISPR/Cas9 for alteration of the kinetochore protein CENH3. TEAM The project is conceived as a collaboration of distinguished scientist with proven track record relevant to this project, belonging to different complementary scientific fields. The exchange of the knowledge, methods and scientific approaches will provide new perspectives and solutions that have not been addressed so far, thus enabling further development of involved scientific fields. This collaboration promises excellent scientific results and technical solutions with possibility of international patent applicatio
Significance for science
The project will combine basic science with a final application in horticulture. The results of the proposed project will have an impact on the implementation of cutting-edge technology in the fields of plant breeding and plant biotechnology and will have a substantial impact on their further development, as we plan to be the first to: 1. Use Cas9-sgRNA ribonucleoprotein complexes (RNPs) for genome editing of any Brassica species. 2. Use RNPs for genome editing of haploid gametes (microspores) of any plant species. 3. Use RNPs in combination with donor DNA for induction of homology-dependent repair (HDR) in plants. 4. Introduce RNPs into plant cells (protoplasts and microspores) by two innovative approaches: with the use of cell penetrating peptides (CPPs) and electroporation. 5. Use CRISPR/Cas9 for development of cabbage plants resistant to Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson. 6. Use CRISPR/Cas9 for alteration of the kinetochore protein CENH3. The project is conceived as a collaboration of distinguished scientist with proven track record relevant to this project, belonging to different complementary scientific fields. The exchange of the knowledge, methods and scientific approaches will provide new perspectives and solutions that have not been addressed so far, thus enabling further development of involved scientific fields. This collaboration promises excellent scientific results and technical solutions with possibility of international patent application.
Significance for the country
The project will combine basic science with a final application in horticulture. The results of the proposed project will have an impact on the implementation of cutting-edge technology in the fields of plant breeding and plant biotechnology and will have a substantial impact on their further development, as we plan to be the first to: 1. Use Cas9-sgRNA ribonucleoprotein complexes (RNPs) for genome editing of any Brassica species. 2. Use RNPs for genome editing of haploid gametes (microspores) of any plant species. 3. Use RNPs in combination with donor DNA for induction of homology-dependent repair (HDR) in plants. 4. Introduce RNPs into plant cells (protoplasts and microspores) by two innovative approaches: with the use of cell penetrating peptides (CPPs) and electroporation. 5. Use CRISPR/Cas9 for development of cabbage plants resistant to Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson. 6. Use CRISPR/Cas9 for alteration of the kinetochore protein CENH3. The project is conceived as a collaboration of distinguished scientist with proven track record relevant to this project, belonging to different complementary scientific fields. The exchange of the knowledge, methods and scientific approaches will provide new perspectives and solutions that have not been addressed so far, thus enabling further development of involved scientific fields. This collaboration promises excellent scientific results and technical solutions with possibility of international patent application.
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