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SCIENCE CHINA Life Sciences, Volume 61, Issue 1: 122-125(2018) https://doi.org/10.1007/s11427-017-9247-9

Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

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  • ReceivedDec 2, 2017
  • AcceptedDec 12, 2017
  • PublishedDec 25, 2017

Abstract

There is no abstract available for this article.


Funded by

the National Key Research and Development Program of China(2017YFD0102002)

the National Natural Science Foundation of China(31271681)

the Zhejiang Provincial Natural Science Foundation of China(LZ14C130003)

and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.


Acknowledgment

This work was supported by the National Key Research and Development Program of China (2016YFD0101800), the National Natural Science Foundation of China (91635301), the Zhejiang Provincial Natural Science Foundation of China (LZ14C130003), and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.


Interest statement

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


Supplement

SUPPORTING INFORMATION

Supplementary Methods

Figure S1 The CRISPR-Cas9 system and sequence of codon-optimized Cas9 used in this study.

Figure S2 The target locations of D1 and D2.

Figure S3 The mutation efficiency of NGG and NAG PAMs.

Figure S4 The assay of mismatch tolerance within the protospacer.

Figure S5 The target locations of NAG1 to NAG8.

Figure S6 Mutational types of rice seedlings with NAG PAMs.

Figure S7 The strategy of mixed PAMs for multiple editing.

Figure S8 The target sites of NGG and NAG in rice genome.

Table S1 The off-target effects with NAG PAM during Bph14 editing

Table S2 The location of double targets of sgRNAs

Table S3 The editing events using sgRNA with mismatches in the protospacer

Table S4 Target sites of AG1–AG8 for genome editing

Table S5 The results of off-target detection

Table S6 Primers used in the study

The supporting information is available online at http://life.scichina.com and https://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

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  • Figure 1

    Genome editing at sites containing NAG PAMs in rice. A, PCR/RE assay of NAG1–NAG8 in protoplasts. Lane 1, undigested wild-type controls. Lane 2, sample treated with Cas9 and sgRNAs. The mutation frequencies measured by band intensities are listed at the bottom. Lane 3, digested wild-type controls. B, Genome editing of NAG1–NAG8 target sites in seedlings. PAMs are highlighted in red and the three bases behind PAMs are exhibited as well. C, The mutational phenotypes of related genes. The wild type (WT) is exhibited on the left. lpa1 shows loose plant architecture. lg1 shows a lack of auricle and ligule. gl1-1nal1 exhibits narrow leaf and loss of cuticular wax. The bar in the image of whole plants represents 10 cm and the bars in images of local features represent 1 cm. D, The sequence of part of mutations. PAMs and targets are highlighted in red and blue, respectively. E, Multiplex editing of V1 and V2. Each column represents one seedling. Blue rectangles indicate target sites with mutations. The numbers on the top indicate the number of co-edited sites and the frequency.

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