DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Journal name:
Nature Biotechnology
Volume:
33,
Pages:
1162–1164
Year published:
DOI:
doi:10.1038/nbt.3389
Received
Accepted
Published online

Abstract

Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

At a glance

Figures

  1. RGEN RNP-mediated gene disruption in plant protoplasts of Nicotiana attenuata, Arabidopsis thaliana and Oryza sativa.
    Figure 1: RGEN RNP-mediated gene disruption in plant protoplasts of Nicotiana attenuata, Arabidopsis thaliana and Oryza sativa.

    (a) Mutation frequencies measured by the T7E1 assay and targeted deep sequencing. “−” indicates controls without RGEN RNP. (b) Mutant DNA sequences induced by RGEN RNPs in plant cells. The PAM sequences are shown in red and inserted nucleotides in blue. WT, wild-type. (c) A time-course analysis of genome editing of the BRI1 gene in A. thaliana protoplasts. Top, the T7E1 assay (“−” indicates controls without Cas9 protein). Bottom, DNA sequences of the wild-type (WT) and mutant sequences.

  2. Targeted gene knockout in lettuce using an RGEN RNP.
    Figure 2: Targeted gene knockout in lettuce using an RGEN RNP.

    (a) The target sequence in the BIN2 gene. The PAM sequence is shown in red. (b) Genotyping of microcalli. Top, RGEN-RFLP analysis. Bottom, mutant DNA sequences in microcalli. (c) Whole plants regenerated from RGEN RNP-transfected protoplasts. Scale bars, 10 cm. (d) T1 plantlets obtained from a homozygous biallelic mutant termed T0-12. Scale bars, 1 cm. (e) RGEN-RFLP analysis for genotyping T1 plantlets. (f) DNA sequences of the wild type, the T0-12 mutant, and T1 mutants derived from the T0-12 line. Red triangles indicate an inserted nucleotide.

  3. Analysis of off-target effects.
    Supplementary Fig. 1: Analysis of off-target effects.

    Mutation frequencies at on-target and potential off-target sites of the PHYB and BRI1 gene-specific sgRNAs were measured by targeted deep sequencing. About ~80,000 paired-end reads per site were obtained to calculate the indel rate.

  4. Partial nucleotide and amino acid sequences of LsBIN2.
    Supplementary Fig. 2: Partial nucleotide and amino acid sequences of LsBIN2.

    Underscored and boxed letters represent the sequences corresponding to degenerate primers and sgRNA, respectively.

  5. Regeneration of plantlets from RGEN RNP-transfected protoplast in L. sativa.
    Supplementary Fig. 3: Regeneration of plantlets from RGEN RNP-transfected protoplast in L. sativa.

    Protoplast division, callus formation and shoot regeneration from RGEN RNP-transfected protoplasts in the lettuce. (a) Cell division after 5 days of protoplast culture (Bar = 100 μm). (b) A multicellular colony of protoplast (Bar = 100 μm). (c) Agarose-embedded colonies after 4 weeks of protoplast culture. (d) Callus formation from protoplast-derived colonies (e,f) Organogenesis and regenerated shoots from protoplast-derived calli (bar = 5 mm).

  6. Targeted deep sequencing of mutant calli.
    Supplementary Fig. 4: Targeted deep sequencing of mutant calli.

    Genotypes of the mutant calli were confirmed by Illumina Miseq. Sequence of each allele and the number of sequencing reads were analyzed. (A1), allele1. (A2), allele2.

  7. Plant regeneration from RGEN RNP-transfected protoplasts in L. sativa.
    Supplementary Fig. 5: Plant regeneration from RGEN RNP-transfected protoplasts in L. sativa.

    (a-c) Organogenesis and shoot formation from protoplast-derived calli; wild type (#28), bi-allelic/heterozygote (#24), bi-allelic/homozygote (#30). (d) In vitro shoot proliferation and development. (e) Elongation and growth of shoots in MS culture medium free of PGR. (f) Root induction onto elongated shoots. (g) Acclimatization of plantlets. (h,i) Regenerated whole plants.

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Primary accessions

Sequence Read Archive

References

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Author information

  1. Present address: Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

    • Seung Woo Cho
  2. These authors contributed equally to this work.

    • Je Wook Woo &
    • Jungeun Kim

Affiliations

  1. Convergence Research Center for Functional Plant Products, Advanced Institutes of Convergence Technology, Yeongtong-gu, Suwon-si, Gyeonggi-do, Korea.

    • Je Wook Woo,
    • Soon Il Kwon &
    • Sunghwa Choe
  2. Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.

    • Jungeun Kim,
    • Hyeran Kim,
    • Sang-Gyu Kim,
    • Sang-Tae Kim &
    • Jin-Soo Kim
  3. Department of Chemistry, Seoul National University, Seoul, South Korea.

    • Jungeun Kim,
    • Seung Woo Cho &
    • Jin-Soo Kim
  4. School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, South Korea.

    • Claudia Corvalán &
    • Sunghwa Choe
  5. Plant Genomics and Breeding Institute, Seoul National University, Seoul, South Korea.

    • Sunghwa Choe

Contributions

J.-S.K. and S.C. supervised the research. J.W.W., S.I.K. and C.C. carried out plant regeneration. J.K., S.W.C. H.K., S.-G.K. and S.-T.K. performed mutation analysis.

Competing financial interests

J.-S.K. and S.C. are co-inventors on a patent application covering the genome editing method described in this manuscript.

Corresponding authors

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Analysis of off-target effects. (140 KB)

    Mutation frequencies at on-target and potential off-target sites of the PHYB and BRI1 gene-specific sgRNAs were measured by targeted deep sequencing. About ~80,000 paired-end reads per site were obtained to calculate the indel rate.

  2. Supplementary Figure 2: Partial nucleotide and amino acid sequences of LsBIN2. (161 KB)

    Underscored and boxed letters represent the sequences corresponding to degenerate primers and sgRNA, respectively.

  3. Supplementary Figure 3: Regeneration of plantlets from RGEN RNP-transfected protoplast in L. sativa. (125 KB)

    Protoplast division, callus formation and shoot regeneration from RGEN RNP-transfected protoplasts in the lettuce. (a) Cell division after 5 days of protoplast culture (Bar = 100 μm). (b) A multicellular colony of protoplast (Bar = 100 μm). (c) Agarose-embedded colonies after 4 weeks of protoplast culture. (d) Callus formation from protoplast-derived colonies (e,f) Organogenesis and regenerated shoots from protoplast-derived calli (bar = 5 mm).

  4. Supplementary Figure 4: Targeted deep sequencing of mutant calli. (133 KB)

    Genotypes of the mutant calli were confirmed by Illumina Miseq. Sequence of each allele and the number of sequencing reads were analyzed. (A1), allele1. (A2), allele2.

  5. Supplementary Figure 5: Plant regeneration from RGEN RNP-transfected protoplasts in L. sativa. (191 KB)

    (a-c) Organogenesis and shoot formation from protoplast-derived calli; wild type (#28), bi-allelic/heterozygote (#24), bi-allelic/homozygote (#30). (d) In vitro shoot proliferation and development. (e) Elongation and growth of shoots in MS culture medium free of PGR. (f) Root induction onto elongated shoots. (g) Acclimatization of plantlets. (h,i) Regenerated whole plants.

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  1. Supplementary Text and Figures (1,201 KB)

    Supplementary Figures 1–5 and Supplementary Tables 1–3

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