Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis
Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis
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应用 CRISPR-Cas12a 温度敏感性改善水稻、玉米和拟南芥基因组编辑
DOI：
10.1186/s12915-019-0629-5
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发表时间：
2019-01-31
期刊：
影响因子：
5.4
通讯作者：
Qi, Yiping
中科院分区：
文献类型：
--
作者：
Malzahn, Aimee A.;Tang, Xu;Qi, Yiping
CRISPR-Cas12a(以前的Cpf1)是一种RNA引导的内切酶，具有扩展了基因组编辑功能的独特功能。Cas12a介导的基因组编辑在植物中是温度敏感的，但由于缺乏对植物细胞中Cas12a温度敏感性的全面了解，阻碍了Cas12a核酸酶在植物基因组编辑中的有效应用。我们比较了AsCas12a、FnCas12a和LbCas12a在四种不同温度下的编辑效率和非同源末端连接(NHEJ)修复谱。我们发现AsCas12a对温度更敏感，它需要超过28 °C的温度才能具有高活性。每个Cas12a核酸酶都表现出不同的Indel突变谱，不受温度的影响。首次成功地应用AsCas12a诱变水稻突变体，突变体频率在T0系中达到93%。接下来，我们在双子叶模式植物拟南芥中进行编辑，以前还没有证明过基于Cas12a的基因组编辑。在22 °C下，LbCas12a几乎没有表现出编辑活性，但通过在29 °C下生长转基因植株，LbCas12a的编辑活性得到了挽救。然后，我们使用高温处理来改进Cas12a介导的玉米基因组编辑。通过在28 °C下生长LbCas12a T0玉米品系，我们在T1代获得了频率高达100%的Cas12a编辑突变体。最后，我们证明了在较低的温度下，利用基于dCas12a-SRDX的转录抑制系统在拟南芥中不会取消Cas12a的DNA结合。我们的研究证明了利用高温机制在水稻、拟南芥和玉米中利用Cas12a系统实现高编辑效率，并揭示了Cas12a在植物中的温度敏感性机制。
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. 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.