CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in <italic>Arabidopsis</italic>

Chen Yiyu; Wang Zhiping; Ni Hanwen; Xu Yong; Chen Qijun; Jiang Linjian

doi:10.1007/s11427-017-9021-5

SCIENCE CHINA Life Sciences

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SCIENCE CHINA Life Sciences, Volume 60, Issue 5: 520-523(2017) https://doi.org/10.1007/s11427-017-9021-5

CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis

Chen Yiyu^1,?, Wang Zhiping^2,?, Ni Hanwen¹, Xu Yong³, Chen Qijun^2,*, Jiang Linjian^1,*

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ReceivedFeb 5, 2017
AcceptedFeb 24, 2017
PublishedMar 16, 2017

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Abstract

There is no abstract available for this article.

Funded by

Special Fund for Agro-scientific Research in the Public Interest(201303022)

National Crop Breeding Fund(2016YFD0101804)

National Natural Science Foundation of China(31471785)

Acknowledgment

Acknowledgements This study was supported by the National Crop Breeding Fund (2016YFD0101804), National Natural Science Foundation of China (31471785, 31670371), and Special Fund for Agro-scientific Research in the Public Interest (201303022).

Interest statement

A patent application related to findings of this report was filed to Patent Office in Beijing, China, in which the authors were listed as inventors.

Supplement

Sequence of maize codon-optimized BE3.

Table S1 Primers used in this study.

Table S2 Genotypes of herbicide resistant T2 seedlings of four edited T1 lines.

Supplemental References

The supporting information is available online at life.scichina.com and link.springer.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

[1] Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and Liu, D. (2016). Google Scholar

[2] Programmable editing of a target base in genomic DNA without dou- ble-stranded DNA cleavage. Nature 533, 420–424. Google Scholar

[3] Li J., Sun Y., Du J., Zhao Y., Xia L.. Generation of Targeted Point Mutations in Rice by a Modified CRISPR/Cas9 System. Mol Plant, 2017, 10: 526-529 CrossRef PubMed Google Scholar

[4] Lu Y., Zhu J.K.. Precise Editing of a Target Base in the Rice Genome Using a Modified CRISPR/Cas9 System. Mol Plant, 2017, 10: 523-525 CrossRef PubMed Google Scholar

[5] Ren B., Yan F., Kuang Y., Li N., Zhang D., Lin H., Zhou H.. A CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic variations in rice. Sci China Life Sci, 2017, 60: 516-519 CrossRef PubMed Google Scholar

[6] Wang Z.P., Xing H.L., Dong L., Zhang H.Y., Han C.Y., Wang X.C., Chen Q.J.. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol, 2015, 16: 144 CrossRef PubMed Google Scholar

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Figure 1
Targeted base-editing in ALS gene efficiently creates inheritable herbicide resistance in Arabidopsis. A, Physical map of pHEE901 harboring Zea mays codon-optimized BE3 driven by the egg-cell specific fusion promoter EC1.2en-EC1.1p (EC1fp) and sgRNA gene driven by Pol-III promoter U6-26p. Only the T-DNA region is shown. RB/LB, T-DNA right/left border; E9t, rbcS E9 terminator; Hyg, hygromycin-resistant gene. The cloning method of target sequence is indicated. B, The alignment of the sgRNA with its target gene. Only regions of interest are aligned and displayed. The amino acids corresponding to the target, and the altered amino acids due to the predicted C to T conversions are indicated. Red arrowhead indicates the nicking site on the non-edited strand (sgRNA complementary strand). HR, herbicide resistant. C, Sequencing chromatograms of “deamination window” of four T1 transgenic lines harboring C to T mutations. Note the heterozygous peaks. The corresponding alleles under each chromatogram were revealed by PCR-cloning sequencing. The mutated bases and resulting mutated amino acids are highlighted in red. D, T2 seedlings of edited T1 lines showed herbicide resistance. T2 and wild type seeds were placed on MS agar plates supplemented with tribenuron at 0 or 5 mg L^-1, vernalized at 4°C for two days, and grown under long-day conditions (16 h light/8 h dark) at 22°C for 10 days before being photographed. Only representative #3-T2 seedlings are shown. E, Sequencing chromatogram of one herbicide resistant T2 plants (#1-7 as shown in Table S2, in Supporting Information) showed that the PAM sequence TGG was edited into TGA, a new deamination target for this base-editing system. F, The occurrence of herbicide resistant plants at T2 generation of 10 randomly selected unedited T1 lines. Mixed Arabidopsis seeds (20 seeds from each unedited T1 line) were sown into soil at 200 seeds per 9-cm square pot. Tribenuron at 13.5 g ai ha^-1 was applied to eight-day old plants. Pictures were taken at 10 days after herbicide treatment. G, Conservation of Pro197 in crops. H, C to T conversions generate expected HR mutations at Pro197 in crops.

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