Molecular Therapy - Nucleic Acids The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo  Su-Ru Lin, Hung-Chih Yang, Yi-Ting Kuo, Chun-Jen Liu, Ta-Yu Yang, Ku-Chun Sung, You-Yu Lin, Hurng-Yi Wang, Chih-Chiang Wang, Yueh-Chi Shen, Fang-Yi Wu, Jia-Horng Kao, Ding-Shinn Chen, Pei-Jer Chen  Molecular Therapy - Nucleic Acids  Volume 3, (January 2014) DOI: 10.1038/mtna.2014.38 Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Designs of gRNAs targeting (hepatitis B virus) HBV-specific sequences. (a) Illustration of the gRNA-targeted sequences located in the HBV genome. (b) Designs of the chimeric HBV-specific gRNAs. Molecular Therapy - Nucleic Acids 2014 3, DOI: (10.1038/mtna.2014.38) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Examination of the efficacy of individual (hepatitis B virus) HBV-specific gRNAs in inhibition of HBV protein expression. The HBV-expression vector was cotransfected to Huh7 cells with (a) each individual gRNA-expression vector and the hCas9 vector or (b) each individual gRNA/Cas9 dual expression vector. The lysate was collected after 48 hours and the levels of intracellular HBcAg and HBsAg were analyzed by using Western blot analysis and quantification of the band intensity. (c) Levels of HBsAg in culture supernatant. The gRNA targeting GFP (GFP-T1), backbone plasmid for the construction of gRNA-expression cassette (blunt), and plasmid containing the U6 promoter/gRNA scaffold (vector) served as negative controls. The bands of *HBs and HBs represent glycosylated and nonglycosylated small surface antigens of HBV, respectively. The relative intensity of HBsAg was shown by quantification of both bands of *HBs and HBs normalized to that of Vector in the bottom panel. RI, relative intensity. The RI and percentages of HBsAg expression were calculated from the results of three independent experiments and presented as mean ± SEM. Statistical significance was computed by Student's t-test and indicated as asterisks (*P < 0.05, **P < 0.01). Molecular Therapy - Nucleic Acids 2014 3, DOI: (10.1038/mtna.2014.38) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Suppression of the (hepatitis B virus) HBV protein expression via the multiplex HBV-specific gRNA. The HBV-expression vector was cotransfected to Huh7 cells with the gRNA/Cas9 dual expression vectors. The lysate was collected after 48 hours and the levels of intracellular HBsAg were analyzed using Western blotting as shown by this representative figure. The band intensities were quantified by software ImageJ. The relative HBsAg intensity (HBs R.I.) in the bar graph of the bottom panel was the results of four independent experiments and presented as mean ± SEM. (b) The DNA extracted from the transfected Huh7 cells in panel (a) was analyzed by T7E1 assay for determining the percentage of Indel as shown in the bottom. Molecular Therapy - Nucleic Acids 2014 3, DOI: (10.1038/mtna.2014.38) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Effects of the gRNAs P1 and XCp on different genotypes of (hepatitis B virus) HBV. (a) HBV-expression vector was cotransfected to Huh7 cells with either the gRNA-P1/Cas9 or gRNA-XCp/Cas9 dual expression vector alone or in combination. The lysate was collected 48 hours post-transfection. The levels of intracellular HBsAg were determined by Western blot analysis. The band intensity was measured by software ImageJ and normalized to the intensity of the mock vector (Vector) for each HBV genotype. The HBs R.I. was calculated from the results of three independent experiments and presented as mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). N.S. means no statistical significance. Sequence alignment of gRNAs P1 (b) and XCp (c) in HBV genotypes A, B, and C. The underlined nucleotides indicated the PAM motif for the recognition of Cas9. Molecular Therapy - Nucleic Acids 2014 3, DOI: (10.1038/mtna.2014.38) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 In vivo destruction of intrahepatic (hepatitis B virus) HBV genomes by the HBV-specific CRISPR/Cas9 system. Sera from mice receiving pAAV/HBV 1.2 together with the gRNA/Cas9 dull expression plasmid, vector (n = 4), gRNA P1 (n = 4), or gRNA XCp (n = 4), were collected after hydrodynamic injection. (a) Levels of HBsAg in sera were measured at days 2 and 7 posthydrodynamic injection as shown in the upper and bottom panel, respectively. The results were presented as individual samples with mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). (b) Analysis of intrahepatic HBV DNA by Southern blotting. 0.1 ng of pAAV/HBV 1.2 plasmid was loaded as a positive control. Two samples from each group were analyzed in this experiment. The band intensity was determined by software ImageJ. The numbers in the bottom indicate the relative intensities of the HBV expression plasmids in each sample. (c) Results of T7E1 assay. The percentage of mismatched sequences from two of the gRNA-P1- or gRNA-XCp-treated mice (Indel %) was determined by T7E1 assay. The numbers in the bottom indicate the percentage of Indel. Molecular Therapy - Nucleic Acids 2014 3, DOI: (10.1038/mtna.2014.38) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions