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Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins

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Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system.

Abstract

The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3–17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

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Acknowledgments

This work was carried out with the support of “Plant Molecular Breeding Center of Next Generation Biogreen 21 Program” (Project No. PJ01119203 to G. -J. L., PJ01119201 to S. B.) funded by Rural Development Administration, Republic of Korea. S. B. was supported by a research fund of Hanyang University (HY-2015).

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Authors and Affiliations

  1. Department of Horticultural Science, Chungnam National University, Daejeon, 305-764, South Korea

    Saminathan Subburaj, Sung Jin Chung & Geung-Joo Lee

  2. Department of Chemistry, Seoul National University, Seoul, 151-742, South Korea

    Choongil Lee, Seuk-Min Ryu & Jin-Soo Kim

  3. Department of Chemistry, Hanyang University, Seoul, 133-791, South Korea

    Duk Hyoung Kim & Sangsu Bae

  4. Center for Genome Engineering, Institute for Basic Science, Seoul, 151-742, South Korea

    Choongil Lee, Seuk-Min Ryu & Jin-Soo Kim

  5. Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, 133-791, South Korea

    Sangsu Bae

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  1. Saminathan Subburaj
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  2. Sung Jin Chung
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  3. Choongil Lee
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Correspondence to Sangsu Bae or Geung-Joo Lee.

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Communicated by T. Cardi.

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Subburaj, S., Chung, S.J., Lee, C. et al. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35, 1535–1544 (2016). https://doi.org/10.1007/s00299-016-1937-7

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