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Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA
Open accessAbstract
RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome, and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA, leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent-free method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing.
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In the citing paper
“RNP electroporation exhibits higher on-target editing efficiency and lower off-target mutations than the other delivery methods”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Ramakrishna et al., 2014
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In the citing paper
“A powerful advantage of the CRISPR/Cas9 system is the ability to target any ∼23-bp sequence that contains a PAM motif on either strand of DNA.”
From Delivering CRISPR: a review of the challenges and approaches (2018) · cites this paper as Ramakrishna et al., 2014
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“cell-penetrating peptides (CPP), conjugated with Cas9 and complexed with gRNA enabled efficient gene silencing in human cell lines and disease models featuring fewer off-target effects when compared to plasmid transfection [153,154].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [153]
Experimental — accuracy not yet measuredIn the citing paper
“Ramakrishna et al. demonstrated that conjugating Cas9 and sgRNA with cell-penetrating peptides (CPPs) enhanced intracellular delivery efficiency because of the membrane-disturbed properties of CPPs.[227]”
From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [227]
In the citing paper
“Some work with CPPs and CRISPR/Cas9 was accomplished as early as 2014 by Ramakrishna et al. (2014), who showed separate delivery of CPP–Cas9 and CPP–sgRNA to multiple human cell lines.”
From Delivering CRISPR: a review of the challenges and approaches (2018) · cites this paper as Ramakrishna et al., 2014
In the citing paper
“Ramakrishna et al. developed cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs for gene disruptions of CCR5 locus in human cell lines, including HEK293T, HeLa, NCCIT (a human pluripotent embryonal carcinoma cell line), human dermal fibroblasts and human embryonic stem cells.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [96]
In the citing paper
“Cell-penetrate-peptide-mediated delivery was reported to achieve higher on-to off-target activity ratio compared with plasmid-mediated delivery.53”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [53]
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Metadata
- Authors
- Ramakrishna, S., Kwaku Dad, A.-B., Beloor, J., Gopalappa, R., Lee, S.-K., & Kim, H.
- Affiliations
- Hanyang University (KR)
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2014-04-02
- Volume
- 24
- Issue
- 6
- Pages
- 1020-1027
- ISSN
- 1088-9051 (ISSN-L)
- PMC
- PMC4032848
- OpenAlex
- W2103609218
- Cited by
- 655
- References
- 45
- Citations by year
- 2026: 16 · 2025: 31 · 2024: 41 · 2023: 46 · 2022: 64 · 2021: 76 · 2020: 66 · 2019: 60 · 2018: 77 · 2017: 60 · 2016: 56 · 2015: 50 · 2014: 11
- Type
- posted content
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc
- Locations
- Genome Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- National Research Foundation
- Hanyang University
- Yonsei University
- Korea Basic Science Institute
- National Research Foundation of Korea (2013M3A9B4076544, 2011‐0019357)
- Korea Advanced Institute of Science and Technology
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas9, cell-penetrating peptides, gene disruption, genome editing, guide RNA delivery, off-target mutations, embryonic stem cells, human cell lines
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Humans, Transfection, Genome, Human, Mutagenesis, Site-Directed, RNA Editing, RNA, Guide, Kinetoplastida, Cell Line, Tumor, Nanoparticles, HEK293 Cells, Cell-Penetrating Peptides, CRISPR-Cas Systems
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