Journal: Molecular Therapy. Methods & Clinical Development
Article Title: Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing
doi: 10.1038/mtm.2016.57
Figure Lengend Snippet: Sendai virus incorporating Cas9 and a guide RNA flanked by self-cleaving ribozymes replicates to high titer. ( a ) The negative-sense RNA genome is flanked by virus promoters (the 3’ leader (le), which serves as the genomic promoter, and the 5’ trailer (tr), which serves as the antigenomic promoter). Shown are the Sendai virus genes N (nucleoprotein), P (phosphoprotein), M (matrix), F (fusion protein), HN (attachment protein), and L (large RNA-dependent RNA polymerase). An EGFP-P2A-Cas9 cassette (5.1 kb) was inserted between N and P, and a guide RNA flanked by self-cleaving ribozymes (rbz 1 and 2) (0.2 kb total) was inserted between P and M (see Materials and Methods for further details). The ribozymes are only functional in the positive-sense, or 5’-to-3’, orientation. Genome may be transcribed from 3’ to 5’ into either full length antigenome or individual capped and polyadenylated mRNAs. These mRNAs are produced in a polar transcriptional gradient, with N mRNAs being the most abundant, and L mRNAs being the least abundant. ( b ) The self-cleaving hammerhead ribozyme sequences and structures are shown. The chimeric guide RNA is shown in orange, corresponding to the orange highlight in panel a . Arrows indicate sites of cleavage. ( c ) The self-cleavage activity of the ribozymes was assayed by qRT-PCR as described in Materials and Methods. Error bars represent standard deviation from 3 independent experiments. ( d ) rSeV-Cas9 (WT), or rSeV-Cas9 with both ribozymes mutated to abolish self-cleavage (Mut) (see for mutations), was rescued from plasmid DNA as described in Materials and Methods. As EGFP is only expressed upon conversion of transfected antigenome to genome and subsequent virus mRNA production, rescue efficiency was determined by observing GFP+ cells (rescue events) by flow cytometry at 1–2 days post-transfection (dpt). Error bars represent standard deviation from 3 replicates. ns, not significant. ( e ) BSR-T7 cells were infected at a multiplicity of infection (MOI) of 0.01. The parental SeV has the EGFP reporter but lacks Cas9 and the guide RNA cassette. Error bars represent standard deviation from three replicates. There was no significant difference ( P > 0.05) between WT and Mut at any time point, two-way ANOVA followed by Bonferroni post-tests. ( f ) HEK293 cells in six-well were transfected with 2 ug px330 (from which the FLAG-tagged Cas9 in rSeV-Cas9 was derived) or infected with rSeV-Cas9 at a MOI of 10. Cell lysates were collected 2 days later and processed via SDS-PAGE and Western blot analysis for detection of the FLAG epitope on Cas9. COX IV represents the loading control.
Article Snippet: FLAG-tagged codon-optimized S. pyogenes Cas9 was amplified from px330 6 (Addgene, cat #42230, from Feng Zhang) and inserted into rSeV following the EGFP reporter, linked with a P2A ribosomal skipping sequence (ATNFSLLKQAGDVEENPGP).
Techniques: Virus, Functional Assay, Produced, Activity Assay, Quantitative RT-PCR, Standard Deviation, Plasmid Preparation, Transfection, Flow Cytometry, Infection, Derivative Assay, SDS Page, Western Blot, FLAG-tag, Control
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