Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis
Aimee A. Malzahn,
Xu Tang,
Keunsub Lee,
Qiurong Ren,
Simon Sretenovic,
Yingxiao Zhang,
Hongqiao Chen,
Minjeong Kang,
Yu Bao,
Xuelian Zheng,
Kejun Deng,
Tao Zhang,
Valeria Salcedo,
Kan Wang,
Yong Zhang,
Yiping Qi
Affiliations
Aimee A. Malzahn
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Xu Tang
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Keunsub Lee
Crop Bioengineering Center, Iowa State University
Qiurong Ren
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Simon Sretenovic
Department of Plant Science and Landscape Architecture, University of Maryland
Yingxiao Zhang
Department of Plant Science and Landscape Architecture, University of Maryland
Hongqiao Chen
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Minjeong Kang
Crop Bioengineering Center, Iowa State University
Yu Bao
Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University
Xuelian Zheng
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Kejun Deng
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Tao Zhang
Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University
Valeria Salcedo
Department of Plant Science and Landscape Architecture, University of Maryland
Kan Wang
Crop Bioengineering Center, Iowa State University
Yong Zhang
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Yiping Qi
Department of Plant Science and Landscape Architecture, University of Maryland
Abstract Background CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. Results We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. Conclusion Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.
