Fig. 1: High efficiency Nrip1 gene disruption at 7 loci by SpyCas9/sgRNA RNPs produces variable degrees of NRIP1 protein loss and UCP1 upregulation in murine primary adipocytes.
From: CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease
a Mapping of the sgRNAs M1-M7 targeting various loci of murine Nrip1 coding region which is entirely located in exon 4 (TSS transcription start site, STOP stop codon). b Time-course of SpyCas9 protein degradation detected by Western Blotting in cell lysates at various time points (0–72 h and day 11 after transfection, which is day 6 of differentiation) after electroporation with RNPs of SpyCas9:sgRNA (3:4 μM). Sup denoted supernatant containing SpyCas9 at 0 h. c Sanger sequencing traces of control vs Nrip1 disrupted cells with sgRNA-M6 showing the sgRNA binding site (solid black line), PAM (red), the double-strand-break (denoted as DSB) site (dashed black line) on the sgRNA-M6 targeting locus and the traces downstream of the DSB created by the DNA repair mechanisms (figure created with SnapGene). d Editing efficiency as evaluated with indel percentage 72 h after the transfection of primary preadipocytes (blue) and differentiation to mature primary adipocytes (orange). e Indel distribution of Nrip1 sgRNA-M6 with frameshift indels that are sustained after differentiation. f Nrip1 gene expression detected by RT-PCR in mature adipocytes targeted with the different sgRNAs. g Immunoprecipitation assay for NRIP1 (140 kDa, arrows at right) in mature primary adipocytes on day 6 post differentiation targeted with the different sgRNAs. The total lysate protein amount used in the assay was 250 μg per sample. M denotes molecular weight marker. Dashed lines separate different gels. h Ucp1 expression by RT-PCR in mature adipocytes targeted with the different sgRNAs compared to non-targeted control cells. i Western blot for UCP1 protein (33 kDa) in mature adipocytes on day 6 post differentiation targeted with the different sgRNAs. Lanes 1–8 were loaded with 20 μg of total protein while lane 9 was loaded with 5 μg οf total protein isolated from mouse BAT. NTC non-targeting control. In (d, e, f and h) bars denote mean and error bars denote Mean ± S.E.M. n ≥ 3 biologically independent replicates. Detailed n per condition and molecular weight markers in (b) and (i) are shown in the source data.
