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. 2019 Jan 31;17(1):9.
doi: 10.1186/s12915-019-0629-5.

Application of CRISPR-Cas12a Temperature Sensitivity for Improved Genome Editing in Rice, Maize, and Arabidopsis

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Free PMC article

Application of CRISPR-Cas12a Temperature Sensitivity for Improved Genome Editing in Rice, Maize, and Arabidopsis

Aimee A Malzahn et al. BMC Biol. .
Free PMC article

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.

Keywords: Arabidopsis; CRISPR-Cas12a; Genome editing; Maize; Rice; Temperature.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Cas12a and Cas9 nuclease activities in rice protoplasts under different temperatures: 22 °C, 28 °C, 32 °C, and 37 °C. Percentage of mutations by AsCas12a, FnCas12a, and LbCas12a at OsROC5 (a) and OsDEP1 (b), and by SpCas9 at OsPDS (c). GFP-transfected samples were used as controls. Error bars represent standard deviations of two biological replicates
Fig. 2
Fig. 2
Three Cas12a nucleases generate slightly distinct mutation profiles that are unaffected by temperatures. OsDEP1 gene targeted by AsCas12a (a), FnCas12a (b), and LbCas12a (c). PAM is red, crRNA is blue. Error bars represent standard deviations of two biological replicates
Fig. 3
Fig. 3
High-temperature treatment resulted in high-frequency genome mutations by AsCas12a in stably transformed rice. a Table of mutation rates and observed genotypes at OsDEP1 and OsROC5. B, biallelic; M, monoallelic; W, wild type. b Sequences of individual mutated T1 rice plants at the target site. PAM is in red and crRNA in blue
Fig. 4
Fig. 4
Activity of LbCas12a is highly sensitive to temperature in Arabidopsis somatic cells. Examples of RFLP gel of GL2 line #8 (a) and TT4 line #2 (c). Presence of a third band indicates mutations. Mutation percentages of GL2 lines #2, #3, #8, and #9 (b) and TT4 lines #2 and #7 (d; plants grown at 29 °C (blue) or 22 °C (red)). Error bars represent standard deviations of five biological replicates
Fig. 5
Fig. 5
Germline mutagenesis by LbCas12a in Arabidopsis with high-temperature treatment. a A summary of genotyping results from GL2 #7-4, GL2 #7-7, and TT4 #9-7. Ho, homozygous; He, heterozygous; B, biallelic; W, wild type. b Target site sequences of gl2 mutants from parental line #7-4, #7-7, and sequences of tt4 mutants. c Images of gl2 mutant (GL2 #7-4-7) and wild type Arabidopsis (WT)
Fig. 6
Fig. 6
High-temperature regime in maize T0 transgenic plants enables high-frequency mutagenesis by LbCas12a. a Diagram of maize transformation and heat treatment. Transgenic plants are crossed with a wild type inbred B104 pollen donor. B, biallelic; M, monoallelic; W, wild type. b Mutation rates and genotyping results of T1 generation from two maize mutant lines, A842B-2-2 and A842B-5-1
Fig. 7
Fig. 7
Quantitative real-time (qRT)-PCR showing dLbCas12a-mediated transcriptional repression in Arabidopsis at different temperatures. a Diagram of a dCas12a-SRDX repressor targeting PAP1. Relative expression of PAP1 mRNA, normalized to EF1α, for two lines at 16 °C (b), 22 °C (c), and 29 °C (d). Error bars represent standard errors of four biological replicates from dCas12a-SRDX transgenic lines PAP #1 and #2, and three biological replicates from WT control plants

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