Extended Data Fig. 3: Biological assay using CRISPR-off at Flower exon 3 impacts Flower isoform expression.
A) A model depicts CRISPR-off strategy for hFwe exon 3. B) Agarose gel electrophoresis of Bi-sulfite-treated and untreated samples derived from WT stromal cells and two clones of CRISPR-off engineered stromal cells. Stromal cells of Clone 1 were co-transfected with CRISPR-off plasmids dCas9-KRAB, dCAS9-D3A-3L, sgRNA targeting hFwe exon 3. Stromal cells of Clone 2 were co- transfected with CRISPR-off plasmids dCas9-DNMT3A, dCAS9- DNMT3B, sgRNA targeting hFwe exon 3. To confirm DNA methylation, we performed Methylation- specific PCR in samples treated or untreated with bi- sulfite and run the products in agarose gels. C) Co-culture of WT tumor and stromal cells shows binding of LINC01914 to hFwe exon 3 DNA, high hFwe exon 3 DNA methylation, enrichment of splicing machinery and high expression of hFwe-Lose isoforms, in stromal cells. Co-culture of LINC01914 KO tumor cells and hFwe exon 3 CRISPR-off stromal cells show no binding of LINC01914 to hFwe exon 3, high DNA methylation, enrichment of splicing machinery at Flower exon 3, and high expression of Flower Lose.
