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Cited by 237CRISPR and Genetic EngineeringPlant Genetic and Mutation StudiesTransgenic Plants and Applications

The DOI matches its Crossref record; 55 of the 55 references it cites were found in a registry or an index.

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

Subburaj, Saminathan, Chung, Sung Jin, Lee, Choongil, Ryu, Seuk-Min, Kim, Duk Hyoung, Kim, Jin-Soo, Bae, Sangsu, Lee, Geung-Joo·2016·Plant Cell Reports
DOI 10.1007/s00299-016-1937-7
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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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  1. Cocking EC, Peberdy JF (1974) The use protoplasts from fungi and higher plants as genetic systems—a practical handbook. Department of Botany, University of Nottingham, Nottingham

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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
ORCID
Venue
Plant Cell Reports
Publisher
Springer Science and Business Media LLC
Published
2016-01-29
Volume
35
Issue
7
Pages
1535-1544
ISSN
  • 0721-7714 (ISSN-L)
  • 1432-203X
DOI
10.1007/s00299-016-1937-7
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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