A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique

Figures

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  Figure 1. In vitro screening of sgRNAs targeting the murine ACBD3 gene and in vivo delivery into the Cas9 knock-in mice using single-stranded adeno-associated virus serotype 8 vector (ssAAV8). (A) sgRNA design for targeting the mouse ACBD3 locus. (B) ACBD3 indel analysis of mouse NIH/3T3 cells transfected with plasmids expressing sgRNAs along with the plasmid expressing Cas9. The results are shown as the percentage of sequencing reads containing indels at the target site. (C) A schematic diagram showing ssAAV8 vector construction expressing sgRNA1 and sgRNA2 under the U6 promoter. (D) ACBD3 indel analysis of the pancreas from the Cas9 knock-in mice injected with ssAAV8 expressing sg-Cont or sg-ACBD3. (E) Cas9 knock-in mice transduced with ssAAV8 expressing sg-Cont or sg-ACBD3 were infected with CVB3 for 3 days. CVB3 titers in the pancreas of the mice were determined as TCID50 per milligram of the tissue (n = 3). Statistical significance was calculated by an unpaired t-test using GraphPad Prism software. Ns indicates p > 0.05.

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  Figure 2. Reduced CVB3 infection in the pancreas of the ACBD3-deficient mice using scAAV8-expressing sg-ACBD3. (A) Schematic diagram of the scAAV8 vector. Vertical bars represent AAV inverted terminal repeats (ITRs). (B) ACBD3 indel analysis of the pancreas from the Cas9 knock-in mice injected with the scAAV8-expressing sg-Cont or sg-ACBD3. (C) Cas9 knock-in mice transduced with the scAAV8-expressing sg-Cont or sg-ACBD3 were infected with CVB3 for 3 days. CVB3 titers in the pancreas of mice were determined as a TCID50 per milligram of the tissue (n = 3). Statistical significance was calculated by an unpaired t-test using GraphPad Prism software. *** indicates p < 0.0001.

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  Figure 3. Immunohistochemical staining of ACBD3 in the pancreas of the Cas9 knock-in mice. Immunohistochemical staining of the pancreatic tissue was performed using an anti-ACBD3 antibody. Representative images of Cas9 knock-in mice without the scAAV8 injection and CVB3 infection, Cas9 knock-in mice injected with the scAAV8-expressing sg-Cont and infected with CVB3, and Cas9 knock-in mice injected with the scAAV8-expressing sg-ACBD3 and infected with CVB3. Bars, 100 µm.

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  Figure 4. Immunohistochemical staining of the viral protein 3C in the pancreas of the Cas9 knock-in mice. Immunohistochemical staining of the pancreatic tissue was performed using the antiviral protein 3C antibody. Representative images of Cas9 knock-in mice without the scAAV8 injection and CVB3 infection, Cas9 knock-in mice injected with the scAAV8-expressing sg-Cont and infected with CVB3, and Cas9 knock-in mice injected with the scAAV8-expressing sg-ACBD3 and infected with CVB3. Bars, 100 µm.

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  Figure 5. Comparison of the human ACBD3 and mouse ACBD3 gene. (A) Schematic diagram of the intron–exon structure for the human ACBD3 and mouse ACBD3 genes generated by Ensembl browser (www.ensembl.org). (B) Blast homology search between human ACBD3 (Query) and mouse ACBD3 (Sbjct) proteins (blast.ncbi.nlm.nih.gov). (C) Schematic diagram of the functional domains of ACBD3. The target sites of sgRNAs are depicted at the bottom.