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

Aimee A Malzahn,Kan Wang,Tao Zhang,Yong Zhang,Xuelian Zheng,Yu Bao,Hongqiao Chen,Keunsub Lee,Kejun Deng,Xu Tang,Qiurong Ren,Yiping Qi,Valeria Salcedo,Minjeong Kang,Simon Sretenovic,Yingxiao Zhang

doi:10.1186/s12915-019-0629-5

Abstract

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 °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.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.

Highlights

CRISPR-Cas12a is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities
Temperature sensitivity of three Cas12a nucleases in rice cells To investigate temperature sensitivity of AsCas12a, FnCas12a, and LbCas12a, we targeted OsROC5 and OsDEP1 in rice protoplasts at 22 °C, 28 °C, 32 °C, and 37 °C. Both Cas12a and the CRISPR RNA (crRNA) were expressed under the maize ubiquitin promoter and the crRNA was precisely processed by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes [17, 18]
The improvement on editing efficiency at higher temperatures was observed for FnCas12a and LbCas12a, but less prominent when compared to AsCas12a (Fig. 1a, b)

Summary

Introduction

CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. In recent years, these SSNs have been overtaken by CRISPR (clustered regularly interspaced short palindromic repeats) systems with Cas or Cas12a (formerly Cpf1) nucleases that mediate DNA targeting through guide RNAs, which are easy to engineer [2,3,4,5,6,7,8]. CRISPR-Cas12a is an RNA-guided endonuclease that has provided new opportunities in genome editing through its distinct features from the commonly used CRISPR-Cas system. Cas12a, unlike other nucleases such as Cas, is not toxic in some organisms such as Chladymonas [13], which expands the spectrums of organisms that can benefit from genome editing. BV3L6 (AsCas12a), and all of them have been tested in plants [14,15,16,17,18]

Methods

Results

Conclusion