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SCIENCE CHINA Life Sciences, Volume 60, Issue 5: 516-519(2017) https://doi.org/10.1007/s11427-016-0406-x

A CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic variations in rice

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  • ReceivedDec 16, 2016
  • AcceptedDec 23, 2016
  • PublishedMar 3, 2017

Abstract

There is no abstract available for this article.


Funded by

the Agricultural Science and Technology Innovation Program of The Chinese Academy of Agricultural Sciences to Huanbin Zhou

and Open Project of State Key Laboratory for Biology of Plant Diseases and Insect Pests

Institute of Plant Protection

to Huanbin Zhou and Dawei Zhang.


Acknowledgment

We thank Xiaonan Zhao, Jining Normal University, for technical assistance; Bo Ding and Qiuyin Yang for critically reading the manuscript. This work was supported by the Agricultural Science and Technology Innovation Program of The Chinese Academy of Agricultural Sciences to Huanbin Zhou, and Open Project of State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, to Huanbin Zhou and Dawei Zhang.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Complete DNA sequences of the rice codon-optimized APOBEC1 and UGI genes.

Figure S2 PCR/RE assay of base editing of OsCERK1 using rBE3 in rice protoplasts.

Figure S3 Representative Sanger sequencing chromatograms of rBE3-edited OsSERK1 gene in T0 rice lines.

Figure S4 Targeted base editing of OsSERK2 in rice using the rBE3 system.

Figure S5 Targeted base editing of ipa1 in rice using the rBE3 system.

Table S1 Rice genes selected for editing with the rBE3 and rBE4 systems

Table S2 List of oligonucleotides used in this study

The supporting information is available online at life.scichina.com and www.springerlink.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


References

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  • Figure 1

    Targeted base editing in rice using the rBE3 and rBE4 system. A, Diagram of the rBE3 system mediating specific, guide RNA-programmed C to U conversion. B, Gene constructs of the rBE3 and rBE4 system used to test targeted base editing activity in rice. C, rBE3 mutated cytosines to thymines at sgRNA-targeted locus of endogenous OsCERK1 gene in rice protoplasts. D, Representative Sanger sequencing chromatogram of cloned OsCERK1 allele with three Cytosines changed. E, rBE3 converted endogenous OsSERK1 genes into kinase-dead versionin rice plants through Agrobacterium-mediated transformation. F, Representative Sanger sequencing chromatogram of cloned OsSERK1 allele with two guanines changed. G, 17% of transgenic rice lines showed target base editing events in OsSERK1 without any indels. H, rBE4 induces genetic variation of rice susceptible gene pi-ta at the key position of Serine 918 through Agrobacterium-mediated transformation. I, Representative Sanger sequencing chromatogram of rBE4-edited pi-ta gene in T0 rice line. J, 18.2% of transgenic rice lines showed target base editing events in pi-ta without any indels. K, Summary of base-editing results of rBE3 and rBE4 in T0 trangenic rice lines. −, not available; √, worked in protoplasts. For (C, E and H), the PAM sequences are in green, putative target bases in the activity window in red, and detected nucleotide mutations in blue. For (D, F and I), nucleotide mutations are underlined in sequencing chromatograms.

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