Fig. 6: Scarless exon-dependent selection marker for the identification of regulators of exon inclusion and exclusion.
From: Non-invasive and high-throughput interrogation of exon-specific isoform expression
a, Split–intein–CC-flanked BSD was integrated into FOXP1 exon 18b of HEK293T cells using CRISPR–Cas9. ESCs, embryonic stem cells. b, A coding gene targeting a genome-wide lentiviral CRISPR–Cas9 library was applied and selected with blasticidin S to enrich cells with FOXP1 exon 18b inclusion. NGS analysis was performed on cells with and without selection to identify the CRISPR–Cas9 spacer leading to cell survival. c, The median-weighted reads for all sgRNA targeting after selection with two concentrations of blasticidin S versus before selection. Two MBNL1-targeting sgRNAs highly enriched under both selection conditions are encircled. The assay was performed once with two different blasticidin S concentrations. d, The results of b were confirmed by an independent EXSISERSFOXP1:18bBSD clone, transfected with a new set of MBNL1-targeting sgRNA together with an MBNL2-targeting sgRNA. Representative surviving colonies, selected with increasing blasticidin S concentrations (top to bottom) after co-transfection with MBNL1 and MBNL2 targeting CRISPR–Cas9 components. The safe-harbour AAVS1 locus was targeted (AAVS1-KO control) as a control. Scale bar, 100 μm. Insets show 5× magnification e, Immunoblot analysis of the cells selected in d. f, RT–PCR analysis of the cells selected in b. For e and f, data represent an independent validation of the results of the screening shown in c. #, 18b EXSISERSBSD isoform; $, 18b isoform. Source data are available online.
