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
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.
Overview
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the citing paper
“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]
Citations
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Development and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013) · Trends in Biotechnology · doi:10.1016/j.tibtech.2013.04.004 · Cited by 3,820
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937
Bae, S., Park, J., & Kim, J.-S. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu048 · Cited by 2,339
Self-citationWang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014) · Nature Biotechnology · doi:10.1038/nbt.2969 · Cited by 2,072
Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013) · Nature Biotechnology · doi:10.1038/nbt.2507 · Cited by 1,863
Self-citationKim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J.-S. (2014) · Genome Research · doi:10.1101/gr.171322.113 · Cited by 1,856
Self-citationTargeted genome modification of crop plants using a CRISPR-Cas system
Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J. J., Qiu, J.-L., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2650 · Cited by 1,810
Research works printed but not linked: 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
Cited by
Sorted by citations
CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture
Chen, K., Wang, Y., Zhang, R., Zhang, H., & Gao, C. (2019) · Annual Review of Plant Biology · doi:10.1146/annurev-arplant-050718-100049 · Cited by 1,696
Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes
Svitashev, S. K., Schwartz, C., Lenderts, B., Young, J. K., & Cigan, A. M. (2016) · Nature Communications · doi:10.1038/ncomms13274 · Cited by 841
DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins
Malnoy, M., Viola, R., Jung, M., Koo, O. J., Kim, S., Kim, J., Velasco, R., & Kanchiswamy, C. N. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.01904 · Cited by 766
Self-citationCRISPR/Cas system: recent advances and future prospects for genome editing
Manghwar, H., Lindsey, K., Zhang, X., & Jin, S. (2019) · Trends in Plant Science · doi:10.1016/j.tplants.2019.09.006 · Cited by 529
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020) · Cells · doi:10.3390/cells9071608 · Cited by 457
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
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
