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Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins
Abstract
Key message 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. 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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“However, due to the large size of Cas9 protein (~ 160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA, the effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult [88–91].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [89]
In the citing paper
“The molecular weight of the commonly used Cas9 protein from streptococcus pyogenes is about 160 KD, larger than that of the most proteins [89].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [89]
In the citing paper
“The first obstacle is effective encapsulation of the editing tools in delivery vectors due to the large size and different charge properties of Cas9 protein, mRNA and donor DNA.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [89]
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Metadata
- Authors
- Subburaj, S., Chung, S. J., Lee, C., Ryu, S.-M., Kim, D. H., Kim, J.-S., Bae, S., & Lee, G.-J.
- Affiliations
- Chungnam National University (KR)
- Seoul National University (KR)
- Hanyang University (KR)
- Corresponding author
- Bae, Sangsu
- Lee, Geung-Joo
- Venue
- Plant Cell Reports
- Published
- 2016-01-29
- Volume
- 35
- Issue
- 7
- Pages
- 1535-1544
- ISSN
- 0721-7714 (ISSN-L)
- 1432-203X
- OpenAlex
- W2469880247
- Cited by
- 237
- References
- 55
- Citations by year
- 2026: 11 · 2025: 12 · 2024: 23 · 2023: 28 · 2022: 35 · 2021: 35 · 2020: 29 · 2019: 25 · 2018: 13 · 2017: 14 · 2016: 12
- FWCI
- 11.09
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Plant Cell Reports (publishedVersion)
- PubMed (publishedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Transgenic Plants and Applications
- Keywords
- CRISPR/Cas9, site-directed mutagenesis, Petunia hybrida, protoplast transformation, nitrate reductase, single guide RNA, ribonucleoprotein delivery, mutation efficiency, plant genome editing
- MeSH terms
- Base Sequence, Genetic Engineering, Microscopy, Fluorescence, Models, Genetic, Plant Proteins, Protoplasts, Recombinant Proteins, Ribonucleoproteins, Sequence Homology, Nucleic Acid, Mutagenesis, Site-Directed, Genome, Plant, Petunia, High-Throughput Nucleotide Sequencing, CRISPR-Cas Systems
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