Journal: The Journal of Biological Chemistry

Article Title: BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

doi: 10.1016/j.jbc.2021.101402

Figure Lengend Snippet: BAd-CRISPR inducesefficientknockout of Adipoq, Atgl, Fasn, Plin1,orScd1 in brown adipocytes of adult mice.A–E, mRNA and protein expression in BAT of BAd-CRISPR mice administered 100 μl 1012 vg/ml AAV8-sgRNA targeting Adipoq, Atgl, Fasn, Plin1, Scd1, or control; mRNA expression was normalized to Ppia (n = 3–4). F, immunofluorescence analysis of paraffin-sectioned BAT from BAd-CRISPR Atgl mice stained for DAPI and immunolabeled against ATGL; 600× magnification. The scale bar represents 50 μm. White arrows indicate brown adipocytes that were not mutated. The data shown are from male (Adipoq, Atgl, Fasn, Plin1, and control) and female (Scd1 and control) mice. The data are presented as mean ± SD. ∗ indicates significance at p < 0.05. AAV, adeno-associated virus; Adipoq, adiponectin; ATGL, adipose triglyceride lipase; BAd-CRISPR, brown adipocyte CRISPR; BAT, brown adipose tissue; CRISPR, clustered regularly interspaced short palindromic repeats; FASN, fatty acid synthase; PLIN, perilipin; SCD, stearoyl CoA desaturase; sgRNA, single guide RNA.

Article Snippet: sgRNA design and cloning sgRNAs were designed using CRISPOR or the Synthego CRISPR Design tool ( 36 ).

Techniques: CRISPR, Expressing, Immunofluorescence, Staining, Immunolabeling

Journal: The Journal of Biological Chemistry

Article Title: BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

doi: 10.1016/j.jbc.2021.101402

Figure Lengend Snippet: BAd-CRISPR Atgl,Plin1, andFasn inducible knockouts recapitulate previously described BAT phenotypes.A, BAT weight (mg) of BAd-CRISPR mice administered 100 μl 1012 vg/ml AAV8-sgRNAs for control, Atgl, Plin1, or Fasn (n = 3–4). B, H&E staining of BAT; 200× magnification. The scale bar represents 50 μm. The data shown are from male mice and are presented as mean ± SD. ∗ indicates significance at p < 0.05. AAV, adeno-associated virus; ATGL, adipose triglyceride lipase; BAd-CRISPR, brown adipocyte CRISPR; BAT, brown adipose tissue; CRISPR, clustered regularly interspaced short palindromic repeats; FASN, fatty acid synthase; PLIN, perilipin; sgRNA, single guide RNA.

Article Snippet: sgRNA design and cloning sgRNAs were designed using CRISPOR or the Synthego CRISPR Design tool ( 36 ).

Techniques: CRISPR, Staining

Journal: The Journal of Biological Chemistry

Article Title: BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

doi: 10.1016/j.jbc.2021.101402

Figure Lengend Snippet: BAd-CRISPR enables simultaneous knockout of two or three genes in brown adipocytes.A, freshly dissected BAT from Rosa26-LSL-Cas9 + AAV8-Atgl sgRNA + AAV8-Plin1 sgRNA (BAd-CRISPR Control), BAd-CRISPR Atgl, BAd-CRISPR Plin1, and BAd-CRISPR Atgl + Plin1 mice administered 100 μl 1010 vg/ml of the designated AAV8 (n = 2). B, H&E staining of BAT; 200× magnification. The scale bar represents 50 μm. C, BAd-CRISPR mice were administered 100 μl 1012 vg/ml of a control AAV8-sgRNA or combinations of AAV8-sgRNAs to Atgl, Plin1, and Ucp1 (n = 2–3). The expression of indicated proteins was determined by immunoblot analyses in the lysates from BAT, psWAT, and eWAT. Laminin is included as a loading control. The data shown are from male mice. AAV, adeno-associated virus; ATGL, adipose triglyceride lipase; BAd-CRISPR, brown adipocyte CRISPR; BAT, brown adipose tissue; CRISPR, clustered regularly interspaced short palindromic repeats; eWAT, epididymal white adipose tissue; FASN, fatty acid synthase; PLIN, perilipin; psWAT, posterior subcutaneous white adipose tissue; sgRNA, single guide RNA; UCP1, uncoupling protein 1.

Article Snippet: sgRNA design and cloning sgRNAs were designed using CRISPOR or the Synthego CRISPR Design tool ( 36 ).

Techniques: CRISPR, Knock-Out, Staining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

doi: 10.1016/j.jbc.2021.101402

Figure Lengend Snippet: BAd-CRISPR ablates Ucp1 expression in BAT.A, confocal micrographs of freshly dissected tissues from BAd-CRISPR Ucp1 mice 14 days after 100 μl 1012 vg/ml AAV8-Ucp1 sgRNA injection; 200× magnification; the scale bar represents 50 μm (n = 3). B, mCherry mRNA expression at each timepoint, RNA expression normalized to Ppia (n = 3 mice per timepoint). C, genomic cleavage assay of cDNA isolated from BAT. The red arrows indicate aberrant mutant PCR products. WT band = 320 bp. + or − indicates addition of the T7 endonuclease. D, Sanger sequencing traces of cDNA from 0 or 14 days postinjection. The expected cut site is indicated with a dashed line and the PAM is underlined in red. Below, the sgRNA sequence is shown, and the PAM is underlined and bolded. The purple arrow indicates the forward primer and sequencing direction. E, mRNA expression of Ucp1 at each timepoint, RNA expression normalized to Ppia (n = 3 mice per timepoint). F, UCP1 and adiponectin protein expression at 0, 2, 7, or 14 days post AAV8-Ucp1 sgRNA injection. The data shown are from female mice and are presented as mean ± SD. ∗ indicates significance at p < 0.05. AAV, adeno-associated virus; Adipoq, adiponectin; ATGL, adipose triglyceride lipase; BAd-CRISPR, brown adipocyte CRISPR; BAT, brown adipose tissue; CRISPR, clustered regularly interspaced short palindromic repeats; FASN, fatty acid synthase; PAM, protospacer-adjacent motif; PLIN, perilipin; pmWAT, parametrial white adipose tissue; psWAT, posterior subcutaneous white adipose tissue; sgRNA, single guide RNA; UCP1, uncoupling protein 1.

Article Snippet: sgRNA design and cloning sgRNAs were designed using CRISPOR or the Synthego CRISPR Design tool ( 36 ).

Techniques: CRISPR, Expressing, Injection, RNA Expression, Cleavage Assay, Isolation, Mutagenesis, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

doi: 10.1016/j.jbc.2021.101402

Figure Lengend Snippet: BAd-CRISPR Ucp1 inducible knockout mice defend core body temperature and have elevatedcirculatingFGF21.A, eight- to 10-week-old Rosa26-Cas9 knockin mice were implanted with a telemeter 7 days before injection with 100 μl 1012 vg/ml AAV8-Control sgRNA or AAV8-Ucp1 sgRNA and single-housed at 20 to 21 °C with no enrichment for 14 days after injection. The mice were cold stressed at 5 °C for 24 h and then sacrificed. B, Ucp1 mRNA expression, mRNA normalized to Ppia (n = 4 or 5 mice). C, immunoblot showing UCP1 and Cas9 expression. D and E, body temperature at 20 to 21 °C and 5 °C. F, relative expression of thermogenic markers in BAT, mRNA normalized to Ppia. G, serum FGF21 concentrations in Rosa26-LSL-Cas9 (BAd-CRISPR Control), Ucp1−/−, or BAd-CRISPR Ucp1 mice (n = 3, 4, or 5 mice). The data shown are from female mice. The data are presented as mean ± SD. ∗ indicates significance at p < 0.05. AAV, adeno-associated virus; BAd-CRISPR, brown adipocyte CRISPR; BAT, brown adipose tissue; CRISPR, clustered regularly interspaced short palindromic repeats; FGF21, fibroblast growth factor 21; sgRNA, single guide RNA; UCP1, uncoupling protein 1.

Article Snippet: sgRNA design and cloning sgRNAs were designed using CRISPOR or the Synthego CRISPR Design tool ( 36 ).

Techniques: CRISPR, Knock-Out, Knock-In, Injection, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

doi: 10.1016/j.jbc.2021.101402

Figure Lengend Snippet: CRISPR/Cas9 does not lead to observable off-target mutations in BAT of BAd-CRISPR Ucp1 mice.A, off-target sequence mismatches for Ucp1 sgRNA predicted by CRISPOR and the Synthego sgRNA Design tool. The Ucp1 sgRNA sequence is highlighted in yellow and base mismatches are colored red. Percent mutations were calculated using whole genome sequencing data visualized by the Integrative Genome Viewer (IGV) and CRISPResso2. B, indel characterization at the intergenic off-target determined by CRISPResso2. C, genomic cleavage assay at the intergenic off-target. WT band = 140 bp. D, heatmap of BAT gene expression for each off-target gene locus represented in the RNA-Seq dataset for BAd-CRISPR Ucp1 mice. ∗ indicates significance at p < 0.05. BAd-CRISPR, brown adipocyte CRISPR; BAT, brown adipose tissue; CRISPR, clustered regularly interspaced short palindromic repeats; sgRNA, single guide RNA; UCP1, uncoupling protein 1.

Article Snippet: sgRNA design and cloning sgRNAs were designed using CRISPOR or the Synthego CRISPR Design tool ( 36 ).

Techniques: CRISPR, Sequencing, Cleavage Assay, Expressing, RNA Sequencing Assay