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SCIENCE CHINA Life Sciences, Volume 62, Issue 7: 982-984(2019) https://doi.org/10.1007/s11427-019-9514-9

Increasing the efficiency of CRISPR/Cas9-based gene editing by suppressing RNAi in plants

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  • ReceivedFeb 19, 2019
  • AcceptedMar 4, 2019
  • PublishedMar 11, 2019

Abstract

There is no abstract available for this article.


Funded by

the Shenzhen Science and Technology Innovation Committee(JCYJ20170818100038326)

Natural Science Foundation of Guangdong Province(2018A030313966)

National Natural Science Foundation of China(31870287)

Guangdong Innovation Team Project(2014ZT05S078)


Acknowledgment

We thank Dr. Qijun Chen for sharing the plasmids pHEE401 and pCBC-DT1T2, Dr. Donald P. Weeks for sharing the vector pCAMBIA-Cas9. This work was supported by the Shenzhen Science and Technology Innovation Committee (JCYJ20170818100038326), Natural Science Foundation of Guangdong Province (2018A030313966), National Natural Science Foundation of China (31870287) and Guangdong Innovation Team Project (2014ZT05S078).


Interest statement

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


Supplement

SUPPORTING INFORMATION

Figure S1 Sequencing analysis of GL1 mutations of representative T1 transgenic plants.

Figure S2 Analysis of CRISPR-targeted GL1 and TRY/CPC mutagenesis in T2 populations.

Figure S3 Characterization of homozygous mutants in T2 populations of amiR-RDR6 CRISPR-GL1.

Table S1 Primers for qRT-PCR and plasmid construction, and amiR-RDR6 sequence

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

    Analysis of CRISPR/Cas9 gene editing efficiency in different genetic backgrounds. A, Physical map of the CRISPR/Cas9 T-DNA vector. sgRNA, single-guide RNA; 35SP, cauliflower mosaic virus 35S promoter; NOST, NOS terminator; U6-26P, U6-26 promoter; U6-26T, U6-26 terminator; U6-29P, U6-29 promoter; HygR, hygromycin resistance. B, Representative phenotypes of strong and moderate CRISPR-targeted transgenic plants. Scale bar, 1.5 cm. C, Phenotype analysis of CRISPR-targeted transgenic plants in different backgrounds. % mutation=(strong+moderate)/total. D, Number of trichomes on the adaxial side of the third and fourth leaves of 4-week-old Arabidopsis plants. Different letters indicate statistically significant differences (ANOVA test, P<0.05). E, Comparison of FLAG-Cas9 expression in different genotypes by Western blot. The black triangle indicates the predicted molecular weight of FLAG-Cas9 protein. The stars indicate the silencing of FLAG-Cas9 expression. HSC70 was used as a loading control. F, Analysis of EC1.2-triggered Cas9 gene editing efficiencies in different genetic backgrounds. G, Phenotypes of 20-day-old amiR-RDR6 transgenic plants. H, qRT-PCR detection of RDR6 transcript levels in amiR-RDR6 transgenic T1 plants. RDR6 transcript levels were normalized to ACT11, and the expression of Col-0 was arbitrarily set to 1. Values are means±SD. I, Phenotype analysis of T2 populations of amiR-RDR6 CRISPR-GL1. The numbers in red indicate putative Cas9-free homozygous mutants. % mutation=(strong+moderate)/total. J, Workflow of the amiR-RDR6 CRISPR/Cas9 system.

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