After the visual novel has been completed at least once, players can view CG (computer graphics) artwork they have observed, skip to scenes or endings for characters they have viewed, and listen to music they have heard in the game.
The simulations started in step 0, where we used the experimentally observed skipping rates (Fig. 3a, black line) as the intended skipping rates (light blue).
We observed skipping with the total AON concentration of 200 nM as well as 100 nM (not shown).
The following alternative splicing events were observed: skipping of exon 2 leading to frameshift and generation of a stop codon in exon 3; skipping of exon 6 (in frame); skipping of exon 12 through 15 with the concomitant inclusion of an additional sequence [GenBank: AM114539] that does not show homology to any of the splice variants reported in other species.
By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves.
The relationship of estimated versus observed skip length had a coefficient of determination of 0.97, which was confirmed by the value of the enhanced Wilmott concordance coefficient of 0.92, indicating good agreement.
DOI: http://dx.doi.org/10.7554/eLife.07540.015 We observed that skipped exons that did not produce circular RNA were significantly shorter compared to the sizes of the predicted circularized exons in S. pombe.
When we transfected the three- or four-antisense PMO cocktails into the MyoD-transduced fibroblasts, we did not observe the skipped products (231 bp) of exons 6-8 on RT-PCR analyses of CXMDJ but did observe the skipped products (99 bp) of exons 6-9.
We also observed heterogenous skipping patterns throughout the muscles which seem to be specific for Mstn and not Dmd.
As anticipated, we observed exon skipping in HCT116 and RPE1 cells in which hCDC14A was targeted by CRISPR-Cas9 (Fig. 4, Additional file 2: Figure S2).
