Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants
Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructi...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2022-01-01
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| Series: | Frontiers in Genome Editing |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fgeed.2021.760820/full |
| _version_ | 1798020439447961600 |
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| author | Raviraj Banakar Raviraj Banakar Raviraj Banakar Mollie Schubert Gavin Kurgan Krishan Mohan Rai Krishan Mohan Rai Krishan Mohan Rai Sarah F. Beaudoin Michael A. Collingwood Christopher A. Vakulskas Kan Wang Kan Wang Feng Zhang Feng Zhang Feng Zhang |
| author_facet | Raviraj Banakar Raviraj Banakar Raviraj Banakar Mollie Schubert Gavin Kurgan Krishan Mohan Rai Krishan Mohan Rai Krishan Mohan Rai Sarah F. Beaudoin Michael A. Collingwood Christopher A. Vakulskas Kan Wang Kan Wang Feng Zhang Feng Zhang Feng Zhang |
| author_sort | Raviraj Banakar |
| collection | DOAJ |
| description | Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants. |
| first_indexed | 2024-04-11T16:57:31Z |
| format | Article |
| id | doaj.art-18bff289bbd546329b3e1e0d0936c3a5 |
| institution | Directory Open Access Journal |
| issn | 2673-3439 |
| language | English |
| last_indexed | 2024-04-11T16:57:31Z |
| publishDate | 2022-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genome Editing |
| spelling | doaj.art-18bff289bbd546329b3e1e0d0936c3a52022-12-22T04:13:14ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392022-01-01310.3389/fgeed.2021.760820760820Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in PlantsRaviraj Banakar0Raviraj Banakar1Raviraj Banakar2Mollie Schubert3Gavin Kurgan4Krishan Mohan Rai5Krishan Mohan Rai6Krishan Mohan Rai7Sarah F. Beaudoin8Michael A. Collingwood9Christopher A. Vakulskas10Kan Wang11Kan Wang12Feng Zhang13Feng Zhang14Feng Zhang15Department of Plant and Microbial Biology, University of Minnesota, St. Paul, MN, United StatesCenter for Precision Plant Genomics, University of Minnesota, St. Paul, MN, United StatesCenter for Genome Engineering, University of Minnesota, St. Paul, MN, United StatesIntegrated DNA Technologies, Coralville, IA, United StatesIntegrated DNA Technologies, Coralville, IA, United StatesDepartment of Plant and Microbial Biology, University of Minnesota, St. Paul, MN, United StatesCenter for Precision Plant Genomics, University of Minnesota, St. Paul, MN, United StatesCenter for Genome Engineering, University of Minnesota, St. Paul, MN, United StatesIntegrated DNA Technologies, Coralville, IA, United StatesIntegrated DNA Technologies, Coralville, IA, United StatesIntegrated DNA Technologies, Coralville, IA, United StatesDepartment of Agronomy, Iowa State University, Ames, IA, United StatesCrop Bioengineering Center, Iowa State University, Ames, IA, United StatesDepartment of Plant and Microbial Biology, University of Minnesota, St. Paul, MN, United StatesCenter for Precision Plant Genomics, University of Minnesota, St. Paul, MN, United StatesCenter for Genome Engineering, University of Minnesota, St. Paul, MN, United StatesDelivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.https://www.frontiersin.org/articles/10.3389/fgeed.2021.760820/fullNicotiana benthamianapennycressprotoplastribonucleoproteinSetaria viridissoybean |
| spellingShingle | Raviraj Banakar Raviraj Banakar Raviraj Banakar Mollie Schubert Gavin Kurgan Krishan Mohan Rai Krishan Mohan Rai Krishan Mohan Rai Sarah F. Beaudoin Michael A. Collingwood Christopher A. Vakulskas Kan Wang Kan Wang Feng Zhang Feng Zhang Feng Zhang Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants Frontiers in Genome Editing Nicotiana benthamiana pennycress protoplast ribonucleoprotein Setaria viridis soybean |
| title | Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants |
| title_full | Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants |
| title_fullStr | Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants |
| title_full_unstemmed | Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants |
| title_short | Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants |
| title_sort | efficiency specificity and temperature sensitivity of cas9 and cas12a rnps for dna free genome editing in plants |
| topic | Nicotiana benthamiana pennycress protoplast ribonucleoprotein Setaria viridis soybean |
| url | https://www.frontiersin.org/articles/10.3389/fgeed.2021.760820/full |
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