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CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins

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Abstract

Main Conclusion

RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology.

Abstract

The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism—knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.

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Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeats

DCL:

Dicer-like

PAM:

Protospacer adjacent motif

RDR6:

RNA-dependent RNA polymerase 6

sgRNA:

Short guide RNA

siRNA:

Small interfering RNA

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Acknowledgements

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant no. 16K14833). We thank Ms. Chihiro Misaki and Ms. Misaki Ito for their careful and skillful assistance in our experiments.

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Correspondence to Kouki Matsuo.

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Fig. S1 Off-target analysis of ΔRDR6 plants. The PAM and the potential off-target sites matching the 12 bp sequence of the target sequence are indicated by red and blue lines, respectively.

Fig. S2 Small RNAs mapped to the GFP open reading frame of WT (W1) and ΔRDR6 plants (A1, A5, A6 and A8).

Fig. S3 Small RNAs (18–50 nt) mapped to the GFP open reading frame of WT (W1) and ΔRDR6 plants (A1, A5, A6 and A8).

Fig. S4 Mapped reads on the GFP open reading frame of WT (W1) and ΔRDR6 plants (A1, A5, A6 and A8).

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Matsuo, K., Atsumi, G. CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins. Planta 250, 463–473 (2019). https://doi.org/10.1007/s00425-019-03180-9

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  • DOI: https://doi.org/10.1007/s00425-019-03180-9

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