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

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.

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Figure 1: RGEN RNP-mediated gene disruption in plant protoplasts of Nicotiana attenuata, Arabidopsis thaliana and Oryza sativa.
Figure 2: Targeted gene knockout in lettuce using an RGEN RNP.

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Acknowledgements

This work was supported in part by grants from the Institute for Basic Science (IBS-R021-D1) and the Next-Generation BioGreen21 Program (PJ01104501 to S.C. and PJ01104502 to S.I.K.).

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Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Sunghwa Choe or Jin-Soo Kim.

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Competing interests

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

Integrated supplementary information

Supplementary Figure 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.

Supplementary Figure 2 Partial nucleotide and amino acid sequences of LsBIN2.

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

Supplementary Figure 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).

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 1173 kb)

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Woo, J., Kim, J., Kwon, S. et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33, 1162–1164 (2015). https://doi.org/10.1038/nbt.3389

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  • DOI: https://doi.org/10.1038/nbt.3389

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