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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Virology. 2014 Dec 29;476:196–205. doi: 10.1016/j.virol.2014.12.001

Figure 1. HBV targeting strategy and sgRNA optimization.

Figure 1

To target HBV DNA intermediates in infected cells, we designed sgRNAs specific for the HBV surface Ag, core, and RT ORFs, as depicted in panel A. To assess efficacy, fusion protein-based indicator plasmids, which encode an amino-terminal HIV-1 Rev-derived epitope tag, an in-frame HBV-derived target, and a carboxy-terminal FLuc indicator gene, were employed. As shown in panel B, all three sgRNA candidates effectively inhibited FLuc expression from their cognate reporter plasmid in co-transfected 293T cells when compared to a control, non-specific (N.S.) sgRNA. In panel C, expression of the same fusion protein was probed by Western blot using a previously described α-Rev rabbit polyclonal antiserum (Kennedy et al., 2014). Co-expression of Cas9 was confirmed using an antibody specific for the FLAG epitope tag present on this protein. When the reporter was cognate for the sgRNA, a marked reduction of the expression level of the fusion protein target could be observed, confirming the specificity and efficacy of each sgRNA tested.