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SCIENCE CHINA Life Sciences, Volume 60, Issue 5: 506-515(2017) https://doi.org/10.1007/s11427-017-9008-8

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

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  • ReceivedDec 1, 2016
  • AcceptedDec 20, 2016
  • PublishedMar 24, 2017

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.


Funded by

National Natural Science Foundation of China(31271681,3140101312)

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

Jiangsu Agriculture Science and Technology Innovation Fund(CX(135075)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (31271681, 3140101312), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and Jiangsu Agriculture Science and Technology Innovation Fund (CX(13)5075).


Open access

This article is distributed under the terms of the Creative Commons Attribution License, which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


Interest statement

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


Supplement

SUPPORTING INFORMATION

Figure S1 Mutation types at the eight target sites in the T0 generation.

Table S1 Mutations detected in putative CRISPR/Cas9 off-target sites

Table S2 Primers 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.


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  • Figure 1

    Schematic diagram of the targeted sites in eight genes. A–H, The targeted sites are labeled in black uppercase letters. The initiation codons are underlined twice. The protospacer adjacent motif (PAM) sequences are underlined once. The arrows show the regions around the editing sites.

  • Figure 2

    Flow diagram of an octuple CRISPR/Cas9 system for multiplex gene editing in rice. The restriction sites used for cloning are labeled. BamH I+Bgl II, Nhe I+Xba I, and Sal I+Xho I are isocaudamer pairs and are highlighted in blue, green, and yellow, respectively.

  • Figure 3

    PCR/RE assay of mutations at eight loci in rice protoplast. BADH2, DEP1, Gn1a, GW2, Hd1, EP3, LPA1, and GS3 products were digested with MscI, NarI, MscI, T7E1, T7E1, HinfI, T7EI, and T7EI, respectively. Lane M, DNA marker. Lane 1, results of PCR/RE assay co-transformed in the single sgRNA (SK-gRNA-BADH2 SK-gRNA-DEP1 SK-gRNA-Gn1a SK-gRNA-GW2 SK-gRNA-Hd1 SK-gRNA-EP3 SK-gRNA-LPA1, and SK-gRNA-GS3, respectively) and Cas9 protein using the transient expression system in rice protoplast. Lane 2, results of PCR/RE assay co-transformed in the four sgRNAs (SK-gRNA-DEP1-Gn1a-GW2-EP3 and SK-gRNA-BADH2-LPA1-GS3-Hd1, respectively) and Cas9 protein using the transient expression system in rice protoplast. Lane 3, results of PCR/RE assay co-transformed in the eight sgRNAs (SK-gRNA-DEP1-Gn1a-GW2-EP3-BADH2-LPA-GS3-Hd1) and Cas9 protein using the transient expression system in rice protoplast. Lane 4, results of PCR/RE assay co-transformed in the control sgRNA (SK-gRNA) and Cas9 protein by using the transient expression system in rice protoplast.

  • Figure 4

    Characterization of targeted editing in T0 rice plants. A, Editing efficiencies of eight agronomic genes in T0 plants. B, Editing efficiencies of wild type (WT), homozygous mutations, and heterozygous mutations in each gene. C, Numbers of plants with different mutation genes. D, Editing efficiencies of off-target genes in T0 plants.

  • Figure 5

    Comparison of panicle traits among Nipponbare (NIP), Mutant 1, and Mutant 2. A, The morphology of the panicles of the NIP, Mutant 1, and Mutant 2. Scale bar, 1 cm. B, Comparison of panicle length among NIP, Mutant 1, and Mutant 2. C, Comparison of grain number per panicle among NIP, Mutant 1, and Mutant 2. Values in B and C are means±standard deviations (SD), n=5. The genotype of mutant-1 and mutant-2 are aabbccddeeffgghh and AABBccDdEEFFGGHH. The letters (a–h), represent BADH2, DEP1, Gn1a, GS3, GW2, Hd1, EP3, LPA1, respectively.

  • Figure 6

    Comparison of seed size among Nipponbare (NIP), Mutant 3, and Mutant 4. Aand B, Grain shape of the NIP, Mutant 3, and Mutant 4. Scale bar, 1 cm. C, Comparison of grain length among NIP, Mutant 3, and Mutant 4. D, Domparison of grain width among NIP, Mutant 3, and Mutant 4. E, Comparison of 1,000-grain weight among NIP, Mutant 3, and Mutant 4. Values in C, D, and F are means±standard deviation (s.d.), n=20, and three replicates. The genotype of mutant-3 and mutant-4 are aabbccddeeffgghh and aaBBccddEEFFGgHh. The letters (a–h), represent BADH2, DEP1, Gn1a, GS3, GW2, Hd1, EP3, LPA1, respectively.

  • Figure 7

    Phenotypes of plants with different gene combinations. A–F, Nipponbare (NIP). The letters (a–h), up from the plants represent BADH2, DEP1, Gn1a, GS3, GW2, Hd1, EP3, LPA1, respectively. Lowercase letters represent the modifed genes, whereas the uppercase letters indicate the normal genes. Scale bar, 10 cm.

  • Table 1   Combination patterns of eight agronomic genes

    Type of gene mutation

    Genotype

    No. of plants

    Sum

    Double mutations

    AABBccDdEEFFGGHH

    3

    3

    Quintuple mutations

    AabbccddEEffGGHH

    2

    8

    aaBbCcddEEffGGHH

    1

    aaBBccddEEFFGgHh

    2

    AABbccddEEffGgHH

    1

    AABbccddEEffGGHh

    1

    AABBCcDdeeffGGHh

    1

    Sextuple mutations

    aabbCcddeeffGGHH

    1

    6

    aaBbCcddeeffGGHH

    2

    aabbccddeeFfGGHH

    1

    aabbccddeeffGGHH

    1

    AaBbCCddEeFfGgHH

    1

    Septuple mutations

    aabbccddEEffGgHh

    1

    10

    aaBbccddEEFfGgHh

    1

    aabbccddeeFFGghh

    2

    aaBbccddEeFFGgHh

    1

    aabbccddeeFfGGhh

    1

    aabbccddeeffGGhh

    1

    aaBbccddeeFfGGhh

    2

    aabbccddeeffGGHh

    1

    Octuple mutations

    AaBbccddEeFfGgHh

    1

    9

    aabbccddeeFfGghh

    4

    aaBbccddEeFfGgHh

    1

    Aabbccddeeffgghh

    2

    AabbccddeeFfGgHh

    1

    The letters (a–h), represent BADH2, DEP1, Gn1a, GS3, GW2, Hd1, EP3, LPA1, respectively.

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