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PreprintCited by 655CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryRNA regulation and disease

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Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA

Open access
Ramakrishna, Suresh, Kwaku Dad, Abu-Bonsrah, Beloor, Jagadish, Gopalappa, Ramu, Lee, Sang-Kyung, Kim, Hyongbum·2014·Genome Research
DOI 10.1101/gr.171264.113PMC PMC4032848
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Abstract

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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Figures

Figure 1. Schematic representation of (A) Cas9 protein conjugated to a cell-penetrating peptide (CPP) and (B) sgRNA complexed with a CPP. The single-letter codes for amino acids are used ([C] cysteine; [G] glycine; [R] arginine; [L] leucine). (His) Histidine tag; (HA) hemagglutinin tag; (Mal) maleimide; (sgRNA) single-guide RNA.
Figure 1. Schematic representation of (A) Cas9 protein conjugated to a cell-penetrating peptide (CPP) and (B) sgRNA complexed with a CPP. The single-letter codes for amino acids are used ([C] cysteine; [G] glycine; [R] arginine; [L] leucine). (His) Histidine tag; (HA) hemagglutinin tag; (Mal) maleimide; (sgRNA) single-guide RNA.CC BY-NC · PubMed Central
Figure 2. Physiochemical characterization of the sgRNA:9R complex. (A–D) A constant amount of sgRNA was complexed with various amounts of 9R. (A) Electrophoretic mobility shift assay. sgRNA was complexed with 9R at various weight ratios and subjected to electrophoresis. (B) Ethidium bromide (EtBr) exclusion assay. The reduction in EtBr fluorescence of sgRNA by complex formation with 9R at various weight ratios was measured. The relative fluorescence intensity is expressed as the percentage of the fluorescence of the uncomplexed sgRNA–EtBr sample. Data represent the average ± SD (n = 3). (C,D) Particle size distribution (C) and surface charge distribution (D) of the complex at different 9R:sgRNA weight ratios were determined using dynamic light scattering (DLS). Data represent the average ± SD (n = 3). (E) Representative scanning electron microscopic image of sgRNA:9R nanoparticles. Scale bar, 500 nm.
Figure 2. Physiochemical characterization of the sgRNA:9R complex. (A–D) A constant amount of sgRNA was complexed with various amounts of 9R. (A) Electrophoretic mobility shift assay. sgRNA was complexed with 9R at various weight ratios and subjected to electrophoresis. (B) Ethidium bromide (EtBr) exclusion assay. The reduction in EtBr fluorescence of sgRNA by complex formation with 9R at various weight ratios was measured. The relative fluorescence intensity is expressed as the percentage of the fluorescence of the uncomplexed sgRNA–EtBr sample. Data represent the average ± SD (n = 3). (C,D) Particle size distribution (C) and surface charge distribution (D) of the complex at different 9R:sgRNA weight ratios were determined using dynamic light scattering (DLS). Data represent the average ± SD (n = 3). (E) Representative scanning electron microscopic image of sgRNA:9R nanoparticles. Scale bar, 500 nm.CC BY-NC · PubMed Central
Figure 3. Cas9-m9R and sgRNA:9R-mediated mutagenesis of an episomal target sequence. (A) Schematic representation of the cell-based reporter assay. (B) Representative flow cytometry of reporter-transfected cells sequentially treated with Cas9-m9R and sgRNA:9R three times or those transfected with plasmids encoding Cas9 and sgRNA as the positive control. The cells were analyzed 3 d after the first treatment with Cas9-m9R and sgRNA:9R or transfection with plasmids encoding Cas9 and sgRNA. The percentages of GFP+ cells are indicated.
Figure 3. Cas9-m9R and sgRNA:9R-mediated mutagenesis of an episomal target sequence. (A) Schematic representation of the cell-based reporter assay. (B) Representative flow cytometry of reporter-transfected cells sequentially treated with Cas9-m9R and sgRNA:9R three times or those transfected with plasmids encoding Cas9 and sgRNA as the positive control. The cells were analyzed 3 d after the first treatment with Cas9-m9R and sgRNA:9R or transfection with plasmids encoding Cas9 and sgRNA. The percentages of GFP+ cells are indicated.CC BY-NC · PubMed Central
Figure 4. Endogenous gene disruption through CPP-mediated delivery of Cas9 protein and guide RNA. HEK293T cells were either sequentially or simultaneously treated with Cas9-m9R and sgRNA:9R three times. (A) Mutations detected by the T7E1 assay 3 d after the first treatment. The arrow indicates the expected position of DNA bands cleaved by T7E1. Mutations frequencies (indel [%]) were calculated from the band intensities. (B) DNA sequences of the CCR5 wild-type (WT) and mutant clones. The target sequence complementary to sgRNA is underlined. The protospacer adjacent motif is shown in bold. The cleavage site is indicated by an arrowhead. The column on the right indicates the number of inserted or deleted bases.
Figure 4. Endogenous gene disruption through CPP-mediated delivery of Cas9 protein and guide RNA. HEK293T cells were either sequentially or simultaneously treated with Cas9-m9R and sgRNA:9R three times. (A) Mutations detected by the T7E1 assay 3 d after the first treatment. The arrow indicates the expected position of DNA bands cleaved by T7E1. Mutations frequencies (indel [%]) were calculated from the band intensities. (B) DNA sequences of the CCR5 wild-type (WT) and mutant clones. The target sequence complementary to sgRNA is underlined. The protospacer adjacent motif is shown in bold. The cleavage site is indicated by an arrowhead. The column on the right indicates the number of inserted or deleted bases.CC BY-NC · PubMed Central
Figure 5. Factors affecting the genome editing efficiency of Cas9-m9R and sgRNA:9R. HEK293T cells were sequentially treated with Cas9-m9R and sgRNA:9R targeting the ABCC11 or CCR5 genes under various conditions, and the resulting mutation frequencies were determined using the T7E1 assay. The arrows indicate the expected position of DNA bands cleaved by T7E1. (A) Multiple treatments with Cas9-m9R and sgRNA:9R increase the mutation frequency. (B) The effect of sgRNA:9R ratios on the mutation frequency. The cells were treated twice with 1 μM Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at various weight ratios. (C) Increasing the Cas9-m9R concentration increases the mutation frequency. The cells were simultaneously treated with various concentrations of Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at a weight ratio of 1:5; this process was repeated three times. (D) The effect of exposure time on the mutation frequency. The cells were sequentially treated with 1.5 μM Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at a weight ratio of 1:5 three times every day for the indicated exposure time per day. The exposure time was controlled by changing the media. (E) Incubating the cells at 30°C instead of 37°C mildly increases the mutation frequencies. Here, the cells were simultaneously treated three times with 1 μM Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at a weight ratio of 1:5.
Figure 5. Factors affecting the genome editing efficiency of Cas9-m9R and sgRNA:9R. HEK293T cells were sequentially treated with Cas9-m9R and sgRNA:9R targeting the ABCC11 or CCR5 genes under various conditions, and the resulting mutation frequencies were determined using the T7E1 assay. The arrows indicate the expected position of DNA bands cleaved by T7E1. (A) Multiple treatments with Cas9-m9R and sgRNA:9R increase the mutation frequency. (B) The effect of sgRNA:9R ratios on the mutation frequency. The cells were treated twice with 1 μM Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at various weight ratios. (C) Increasing the Cas9-m9R concentration increases the mutation frequency. The cells were simultaneously treated with various concentrations of Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at a weight ratio of 1:5; this process was repeated three times. (D) The effect of exposure time on the mutation frequency. The cells were sequentially treated with 1.5 μM Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at a weight ratio of 1:5 three times every day for the indicated exposure time per day. The exposure time was controlled by changing the media. (E) Incubating the cells at 30°C instead of 37°C mildly increases the mutation frequencies. Here, the cells were simultaneously treated three times with 1 μM Cas9-m9R and 10 μg/mL sgRNA complexed with 9R at a weight ratio of 1:5.CC BY-NC · PubMed Central
Figure 6. Endogenous gene disruption through CPP-mediated delivery of Cas9 protein and guide RNA in several cell lines. Cell lines such as HEK293T (human embryonic kidney cells), HeLa (human cervical cancer cell line), and NCCIT (human embryonal carcinoma cell line) cells (A), and primary cells such as human dermal fibroblasts (HDF) and human embryonic stem cells (B), were treated with Cas9-m9R and sgRNA:9R once per day for 3 d. The gene disruption efficiencies were determined using the T7E1 assay 3 d after the first treatment. The arrow indicates the expected position of DNA bands cleaved by T7E1.
Figure 6. Endogenous gene disruption through CPP-mediated delivery of Cas9 protein and guide RNA in several cell lines. Cell lines such as HEK293T (human embryonic kidney cells), HeLa (human cervical cancer cell line), and NCCIT (human embryonal carcinoma cell line) cells (A), and primary cells such as human dermal fibroblasts (HDF) and human embryonic stem cells (B), were treated with Cas9-m9R and sgRNA:9R once per day for 3 d. The gene disruption efficiencies were determined using the T7E1 assay 3 d after the first treatment. The arrow indicates the expected position of DNA bands cleaved by T7E1.CC BY-NC · PubMed Central
Figure 7. Reduced off-target effects of Cas9-m9R and sgRNA:9R. HEK293T cells were treated with Cas9-m9R and sgRNA:9R once per day for 3 d. Three days after the first treatment, the mutation frequencies at on- and off-target sites were determined using the T7E1 assay. The arrows indicate the expected position of DNA bands cleaved by T7E1. The protospacer adjacent motif is shown in bold. Mismatched bases are underlined.
Figure 7. Reduced off-target effects of Cas9-m9R and sgRNA:9R. HEK293T cells were treated with Cas9-m9R and sgRNA:9R once per day for 3 d. Three days after the first treatment, the mutation frequencies at on- and off-target sites were determined using the T7E1 assay. The arrows indicate the expected position of DNA bands cleaved by T7E1. The protospacer adjacent motif is shown in bold. Mismatched bases are underlined.CC BY-NC · PubMed Central

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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)
ORCID
Venue
Genome Research
Publisher
Cold Spring Harbor Laboratory
Published
2014-04-02
Volume
24
Issue
6
Pages
1020-1027
ISSN
1088-9051 (ISSN-L)
DOI
10.1101/gr.171264.113
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
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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