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Genome editing in plants by engineered CRISPR–Cas9 recognizing NG PAM

Abstract

Streptococcus pyogenes Cas9 (SpCas9) is widely used for genome editing and requires NGG as a protospacer adjacent motif (PAM). Here, we show that the engineered SpCas9 (SpCas9-NGv1) can efficiently mutagenize endogenous target sites with NG PAMs in the rice and Arabidopsis genomes. Furthermore, we demonstrate that the SpCas9-NGv1 nickase fused to cytidine deaminase mediates C-to-T substitutions near the 5′ end of the target sequence.

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The remaining data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The plant CRISPR vector (pDeCas9) harbouring the SpCas9-WT gene was kindly provided by F. Fauser, S. Schiml and H. Puchta, Karlsruhe Institute of Technology (Karlsruhe, Germany). The Target-AID vector (nCas9Os-PmCDA1At) was kindly provided by A. Kondo and K. Nishida, Kobe University (Kobe, Japan). We are grateful to S. Gelvin, Purdue University (West Lafayette, IN, USA), for critical reading of the manuscript and helpful suggestions. Digenome sequencing was accomplished by the support of the Advanced Analysis Center, National Agriculture and Food Research Organization (Tsukuba, Japan). This work was supported by the Cabinet Office, Government of Japan, Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Biooriented Technology Research Advancement Institution, NARO).

Author information

S.T. and O.N. conceived the idea for the study. H.N. conducted the in vitro DNA cleavage assay. M.E., M.M., A.E. and H.K. constructed the plasmids. M.E. and M.M. analysed the rice mutants. A.E. and H.K. analysed the transfected Arabidopsis protoplast. A.E., H.K. and T.I. conducted the Digenome sequencing. M.E. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Seiichi Toki.

Supplementary information

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    Supplementary Text, Supplementary Figures 1–9, Supplementary Tables 1–2 and online methods.

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DOI

https://doi.org/10.1038/s41477-018-0321-8

Further reading

Fig. 1: SpCas9-NGv1-mediated targeted mutagenesis in rice.
Fig. 2: SpCas9-NGv1-mediated C-to-T substitution in rice.