Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis

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dc.contributor.authorMalzahn, Aimee A.
dc.contributor.authorTang, Xu
dc.contributor.authorLee, Keunsub
dc.contributor.authorRen, Qiurong
dc.contributor.authorSretenovic, Simon
dc.contributor.authorZhang, Yingxiao
dc.contributor.authorChen, Hongqiao
dc.contributor.authorKang, Minjeong
dc.contributor.authorBao, Yu
dc.contributor.authorZheng, Xuelian
dc.contributor.authorDeng, Kejun
dc.contributor.authorZhang, Tao
dc.contributor.authorSalcedo, Valeria
dc.contributor.authorWang, Kan
dc.contributor.authorZhang, Yong
dc.contributor.authorQi, Yiping
dc.date.accessioned2021-06-10T19:45:19Z
dc.date.available2021-06-10T19:45:19Z
dc.date.issued2019-01-31
dc.description.abstractCRISPR-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. 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. 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.en_US
dc.description.urihttps://doi.org/10.1186/s12915-019-0629-5
dc.identifierhttps://doi.org/10.13016/g6vi-vcd9
dc.identifier.citationMalzahn, A.A., Tang, X., Lee, K. et al. Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biol 17, 9 (2019).en_US
dc.identifier.urihttp://hdl.handle.net/1903/27150
dc.language.isoen_USen_US
dc.publisherSpringer Natureen_US
dc.relation.isAvailableAtCollege of Agriculture & Natural Resourcesen_us
dc.relation.isAvailableAtPlant Science & Landscape Architectureen_us
dc.relation.isAvailableAtDigital Repository at the University of Marylanden_us
dc.relation.isAvailableAtUniversity of Maryland (College Park, MD)en_us
dc.subjectCRISPR-Cas12aen_US
dc.subjectTemperatureen_US
dc.subjectGenome editingen_US
dc.subjectRiceen_US
dc.subjectArabidopsisen_US
dc.subjectMaizeen_US
dc.titleApplication of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsisen_US
dc.typeArticleen_US

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