Abstract
Main conclusion
A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation.
Abstract
Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation ‘Kane Ainou 1-go’. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of ‘Kane Ainou 1-go’ has not been previously isolated, we initially isolated the ANS gene from ‘Kane Ainou 1-go’ for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.
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Data availability
All data generated during this study are included in this published article and its supplementary information files.
Abbreviations
- ANS:
-
Anthocyanidin synthase
- CAPS:
-
Cleaved amplified polymorphic sequence
- Cas9:
-
CRISPR-associated protein 9
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- RNP:
-
Ribonucleoprotein
- sgRNA:
-
Single guide RNA
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Acknowledgements
We thank Ms. Miyuki Tsuruoka, Ms. Yuko Namekawa, Ms. Yumi Shimura, and Ms. Ayami Aoyama of NARO for their assistance in maintaining the carnation plants and Ms. Yoshie Niwa, Mr. Noriyoshi Chiso, and Ms. Hiroko Toda of AARC for their advice on growing carnation cuttings. We also thank Mr. Toyohisa Kurita of Takara Bio Inc. for providing the Guide-it™ sgRNA In Vitro Transcription Kit and Guide-it™ Recombinant Cas9, as well as Mr. Kojiro Matsumoto of NEPA Gene Inc. for his assistance in using the electroporator NEPA21 Type II.
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This work was supported by NARO.
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KM and KS planned the study. KM mainly performed the analyses, and KS and KT performed other analyses. KM and KS prepared the draft of the manuscript. KS provided essential advice for performing the experiments. KM, KT, and KS reviewed and revised the manuscript.
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425_2024_4358_MOESM1_ESM.pptx
Supplementary file 1—Figure S1. ANS-1 gene and amino acid sequence. A Genomic sequence of ANS-1 (1223 bp). The intron is highlighted in gray. B Amino acid sequence of ANS-1 (360 aa). Fig. S2. ANS-2 gene and amino acid sequence. A Genomic sequence of ANS-2 (1234 bp). The intron is highlighted in gray. B Amino acid sequence of ANS-2 (360 aa). Fig. S3. Alignment data of nucleotide sequences of ANS-1 and ANS-2. Alignment data of genomic sequences of ANS-1 and ANS-2. Lower case letters indicate the intron of ANS-1 and ANS-2. The lower case letters in the sequences indicate the intron region. Fig. S4. Alignment data of amino acid sequences of ANS-1 and ANS-2. Fig. S5. In vitro cleavage assay using CRISPR/Cas9 of the ANS gene with the addition of surfactant SILWET L-77 and depressurization treatment. Amplified DNA fragments of the ANS gene were digested with Cas9. A Results following the addition of surfactant SILWET L-77. B Results after the addition of surfactant SILWET L-77 and depressurization. Lanes marked with red letter numbers indicate lanes where bands of size not cut by PstI were identified. Fig. S6. Details of electrical conditions. Black letters indicate setting values for the poring pulse and transfer pulse. Red letters indicate the measured values for the poring pulse, transfer pulse, and electrical resistance (PPTX 4683 KB)
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Mori, K., Tanase, K. & Sasaki, K. Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene. Planta 259, 84 (2024). https://doi.org/10.1007/s00425-024-04358-6
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DOI: https://doi.org/10.1007/s00425-024-04358-6