Generation of hepatitis C virus–resistant liver cells by genome editing–mediated stable expression of RNA aptamer

Figures

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  Figure 1. Characterization of HCV-specific RNA aptamers and anti-HCV efficiency of RNA aptamer in HCV stable cells (A) Secondary structure of aptamer R16, CH2, and R16_CH2. R16 is an HCV NS5B replicase-specific RNA aptamer. CH2 is an HCV NS3 helicase-specific RNA aptamer. R16_CH2 is a conjugated chimeric aptamer of R16 and CH2. The red color represents the predicted binding region of R16. The yellow color represents the predicted binding region of CH2. (B) Schematic diagram of the SNAP31 RNA expression system with HCV-specific RNA aptamers in a cellular model. (C and D) HCV genotype 1b subgenomic replicon cells (C) or HCV genotype 2a JFH-1 stable cells (D) were transfected with each SNAP31-aptamer construct. HCV RNA was assessed by qRT-PCR and expressed relative to the level in cells transfected with the SNAP31 vector. The results are presented as means ± SDs of triplicate measurements. ∗∗∗p < 0.0005.

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  Figure 2. Characterization of HCV NS5B-specific RNA aptamers and development of aptamer stable cells (A) Secondary structures of R70, R70 ×3, and R70 ×5. R70 is an HCV NS5B-specific RNA aptamer, which was a minimized form of R16. R70 ×3 is a conjugated form of three copies of R70. R70 ×5 is a conjugated form of five copies of R70. The sequences with red color represent the predicted binding region of R70. (B) The expression of RNA aptamer in each developed RNA aptamer stable cell was assessed by RT-PCR and 2% agarose gel electrophoresis. M indicates the DNA size marker. Human 18S rRNA was amplified as the internal control.

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  Figure 3. HCV inhibitory effect of the developed RNA aptamer stable cells (A and B) HCV genotype 1b subgenomic replicon RNA (A) or HCV genotype 2A JFH-1 RNA (B) was transfected into each aptamer stable cell line. (C and D) HCV genotype 2A JFH-1 virus was used for infection at an MOI of 0.1 (C) or 0.5 (D) into each aptamer stable cell line. HCV genomic RNA levels were assessed by qRT-PCR and expressed relative to the level in SNAP31 stable cells. The results are presented as means ± SDs of triplicate measurements. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005. (E) Each aptamer stable cell was transfected with HCV genotype 1b subgenomic replicon RNA and selected with puromycin and G418. Colony formation is shown.

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  Figure 4. Development of AAVS1 HCV-specific RNA aptamer stable cells using the CRISPR-Cas9 system (A) Schematic diagram of the SNAP31 construct encoding an HCV-specific RNA aptamer with a DHFR minigene and knockin of the construct into AAVS1 through HDR. (B) The sequence of AAVS1 sites targeted by sgRNAs (top) and the T7 endonuclease 1 (T7E1) assay with each sgRNA (bottom). (C) The expression of RNA aptamer was assessed by RT-PCR in each AAVS1 HCV-specific RNA aptamer knockin cell clone and 2% agarose gel electrophoresis. The numbers represent clone numbers for each aptamer stable cell. M indicates the DNA size marker. NTC denotes the PCR controls. Human 18S rRNA was amplified as the internal control. (D) Genomic DNA was assessed in each AAVS1 knockin cell clone expressing HCV-specific RNA aptamers by PCR and 1% agarose gel electrophoresis. The numbers represent clone numbers for each aptamer stable cell. The expected band size is indicated by an arrow. M indicates the DNA size marker. NTC denotes PCR controls. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as the internal control.

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  Figure 5. HCV inhibitory effect of developed AAVS1 RNA aptamer knockin cell clones (A–D) HCV genotype 1b subgenomic replicon RNA (A) or HCV genotype 2A JFH-1 RNA (B) was transfected into each AAVS1 aptamer stable cell clone. HCV genotype 2A JFH-1 virus was used for infection at an MOI of 0.1 (C) and 0.5 (D) into each AAVS1 aptamer stable cell clone. HCV genomic RNA level was assessed by qRT-PCR and expressed relative to the level in the AAVS1 SNAP31 knockin cell clone. The results are presented as means ± SDs of triplicate measurements. ∗∗p < 0.005; ∗∗∗p < 0.0005. (E and F) HCV genotype 1b subgenomic replicon RNA (E) or HCV genotype 2A JFH-1 RNA (F) was transfected into each AAVS1 aptamer stable cell clone. The NS5B protein level of each cell clone was measured by immunoblotting. α-Tubulin was immunoblotted as the internal control. WB, western blot.

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  Figure 6. Inhibitory mechanism of RNA aptamer of each AAVS1 knockin cell clone (A and B) HCV genotype 1b subgenomic replicon RNA (A) or HCV genotype 2A JFH-1 RNA (B) was transfected into each AAVS1 aptamer stable cell clone, and RNA IP was performed for HCV RNA-treated cells. NS5B proteins in cell extracts after IP with IgG or NS5B antibody were assessed by immunoblotting (top). RNA was amplified by RT-PCR from input or immunoprecipitate using IgG or NS5B antibody in each cell extract. The expected band size is indicated by an arrow. M indicates the DNA size marker. NTC denotes PCR controls. Lane 1: SNAP31 #1 cell, lane 2: SNAP31-NFATc #8 cell, lane 3: SNAP31-R16 #11 cell, lane 4: SNAP31-R16_CH2 #4 cell, lane 5: SNAP31-R70 #7 cell, lane 6: SNAP31-R70 ×3 #7 cell, lane 7: SNAP31-R70 ×5 #7 cell. (C and D) HCV genotype 1b subgenomic replicon RNA (C) or HCV genotype 2a JFH-1 RNA (D) was cotransfected into each aptamer stable cell line with the HCV NS3 or NS5B expression plasmid. HCV genomic RNA level was assessed by qRT-PCR and expressed relative to the level in SNAP31 stable cells. The results are presented as means ± SDs of triplicate measurements. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005. (E)…

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  Figure 7. Development of AAVS1 knockin cells stably expressing HCV-specific aptamer DSBs in the genome were induced with the CRISPR-Cas9 system targeting AAVS1. HDR occurs by homology of donor constructs to the target, and knockin of the donor constructs occurs. The RNA aptamer is stably expressed in the cytoplasm of the knockin cells. It inhibits HCV replication by targeting HCV viral proteins. PAM, protospacer adjacent motif.