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

被引：175

作者：

Malzahn, Aimee A. ^{[1
,2
]}

Tang, Xu ^{[1
]}

Lee, Keunsub ^{[3
,4
]}

Ren, Qiurong ^{[1
]}

Sretenovic, Simon ^{[2
]}

Zhang, Yingxiao ^{[2
]}

Chen, Hongqiao ^{[1
]}

Kang, Minjeong ^{[3
,4
,5
]}

Bao, Yu ^{[6
,7
]}

Zheng, Xuelian ^{[1
]}

Deng, Kejun ^{[1
]}

Zhang, Tao ^{[6
,7
]}

Salcedo, Valeria ^{[2
]}

Wang, Kan ^{[3
,4
]}

Zhang, Yong ^{[1
]}

Qi, Yiping ^{[2
,8
]}

机构：

[1] Univ Elect Sci & Technol China, Sch Life Sci & Technol, Dept Biotechnol, Ctr Informat Biol, Chengdu 610054, Sichuan, Peoples R China

[2] Univ Maryland, Dept Plant Sci & Landscape Architecture, College Pk, MD 20742 USA

[3] Iowa State Univ, Crop Bioengn Ctr, Ames, IA 50011 USA

[4] Iowa State Univ, Dept Agron, Ames, IA 50011 USA

[5] Iowa State Univ, Interdept Plant Biol Major, Ames, IA 50011 USA

[6] Yangzhou Univ, Key Lab Plant Funct Genom, Coinnovat Ctr Modern Prod Technol Grain Crops, Jiangsu Key Lab Crop Genet & Physiol,Minist Educ, Yangzhou 225009, Jiangsu, Peoples R China

[7] Yangzhou Univ, Joint Int Res Lab Agr & Agriprod Safety, Minist Educ China, Yangzhou 225009, Jiangsu, Peoples R China

[8] Univ Maryland, Inst Biosci & Biotechnol Res, Rockville, MD 20850 USA

来源：

BMC BIOLOGY | 2019年 / 17卷 / 1期

基金：

美国国家科学基金会;

关键词：

CRISPR-Cas12a; Temperature; Genome editing; Rice; Arabidopsis; Maize; TARGETED MUTAGENESIS; WIDE ANALYSIS; CPF1; CRISPR/CAS9; SPECIFICITIES; ENDONUCLEASE; EFFICIENCY; NUCLEASES; SYSTEM; TOOL;

D O I：

10.1186/s12915-019-0629-5

中图分类号：

Q [生物科学];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

BackgroundCRISPR-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.ResultsWe 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 degrees 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 degrees C, its editing activity was rescued by growing the transgenic plants at 29 degrees 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 degrees 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.ConclusionOur 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.

引用

页数：14

共 57 条

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