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. 2015 Sep;21(9):1683–1689. doi: 10.1261/rna.051631.115

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© 2015 Mefferd et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 3. — Various human tRNAs can be used to express S. aureus Cas9-specific sgRNAs. (A) Relative luciferase activity from 293T cells cotransfected with plasmids expressing the Sau Cas9 protein and sgRNAs specific for the HSV-1 ICP0 gene or a no sgRNA control (Neg), and a cognate ICP0-based indicator plasmid. sgRNAs were expressed using the various human tRNAs indicated. Average of three experiments with SD indicated. (B) Northern blot of Sau sgRNAs recovered from 293T cells transfected with a plasmid expressing Sau Cas9 and an sgRNA specific for HSV-1 ICP0. The sgRNAs were expressed using the indicated human tRNAs. Endogenous cellular U6 RNA served as a loading control. The arrow indicates the predicted location of the mature sgRNA, while the asterisk indicates the unprocessed tRNA:sgRNA fusion transcript. The latter varies in size depending on the size of the tRNA used. (C) Similar to B, except that this Northern analyzes the expression of sgRNAs transcribed using human tRNA_GLN1 that are specific for the HSV-1 ICP0 or ICP4 genes or the HBV surface antigen (SA) gene. (D) Northern blot analysis of Sau sgRNAs recovered from 293T cells cotransfected with a plasmid expressing an sgRNA targeting HSV-1 ICP0 from either a U6 or tRNA_GLN1 promoter and a plasmid expressing Sau Cas9 or an empty vector control. Endogenous cellular U6 RNA served as a loading control.