Figure 1.
CRISPR/Cas9–mediated deletion of MIR155HG results in downregulation of miR-155 expression with minimal off-target effects. (A) MIR155HG gene locus with 3 exons in blue shaded boxes and promoter region is shown. Yellow shaded box within exon 3 denotes pre-miR-155. Guide RNAs (gRNAs) targeting promoter region are shown in dark blue, and gRNAs targeting miR-155 sequence in exon 3 junction are shown in green. (B) Genomic PCR performed 72 hours after transfection. One representative donor is shown. (C) Validation of sgRNA pairs to target MIR155HG. Fold change in miR-155 expression measured using quantitative reverse transcriptase–PCR (qRT-PCR) at 72 hours (left) and 7 days after transfection (right). (D) On-target efficiency was quantified by droplet digital PCR performed 72 hours after transfection. (E) qRT-PCR, n = 3 donors and (F) Western blot analysis of identified off-target genes from a single representative donor. (G) Western blot analysis SOCS1 and WEE1 from a single representative donor. (H) Densitometric quantification of panel G, n = 4 donors. Data are combined from 3 to 16 independent donors, each symbol represents an individual donor (∗∗P < .01, ∗∗∗∗P < .0001).

CRISPR/Cas9–mediated deletion of MIR155HG results in downregulation of miR-155 expression with minimal off-target effects. (A) MIR155HG gene locus with 3 exons in blue shaded boxes and promoter region is shown. Yellow shaded box within exon 3 denotes pre-miR-155. Guide RNAs (gRNAs) targeting promoter region are shown in dark blue, and gRNAs targeting miR-155 sequence in exon 3 junction are shown in green. (B) Genomic PCR performed 72 hours after transfection. One representative donor is shown. (C) Validation of sgRNA pairs to target MIR155HG. Fold change in miR-155 expression measured using quantitative reverse transcriptase–PCR (qRT-PCR) at 72 hours (left) and 7 days after transfection (right). (D) On-target efficiency was quantified by droplet digital PCR performed 72 hours after transfection. (E) qRT-PCR, n = 3 donors and (F) Western blot analysis of identified off-target genes from a single representative donor. (G) Western blot analysis SOCS1 and WEE1 from a single representative donor. (H) Densitometric quantification of panel G, n = 4 donors. Data are combined from 3 to 16 independent donors, each symbol represents an individual donor (∗∗P < .01, ∗∗∗∗P < .0001).

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