Journal: Science Advances
Article Title: Modulating immune cell fate and inflammation through CRISPR-mediated DNA methylation editing
doi: 10.1126/sciadv.adt1644
Figure Lengend Snippet: ( A ) Schematic of the dCas9-TET1 IL1RN promoter epigenome editing. The four sgRNAs targeting the IL1RN TSS are shown. #1 to #4 indicate the CpGs analyzed in (C). ( B ) ChIP-qPCR showing dCas9-TET1 enrichment at the IL1RN promoter. A 5-kb downstream region from the TSS is shown as a negative control region. Unpaired two-tailed Student’s t test, n = 3, mean SEM, (**** P < 0.0001). ( C ) DNAm levels (pyrosequencing) at four CpGs upstream of IL1RN TSS [as in (A)] in CTRL and edited B cells. One-way analysis of variance (ANOVA) with Dunnett’s post hoc correction, n = 3, means ± SEM, (* P < 0.05; ** P < 0.01, *** P <<0.001,**** P < 0.0001). ( D ) Top, IL1RN expression (RT-qPCR) in CTRL and edited B cells. Unpaired two-tailed Student’s t test, n = 3, means ± SEM, (*** P < 0.001). Bottom, IL1RN protein levels in CTRL and edited B cells. Fold change normalized to β-actin (CTRL versus sgIL1RN). ( E ) Schematic of the dCas9-DNMT3A IL1RN promoter epigenome editing experiment as in (A), showing two Infinium MethylationEPIC v2.0 probes. ( F ) Genome browser snapshot showing dCas9-DNMT3A binding at the IL1RN promoter. The location of the four sgRNAs targeting the IL1RN promoter and the two array probes is depicted. ( G ) Scatter plot showing differential dCas9-DNMT3A enrichment in sgIL1RN B cells. Large dots indicate top-bound promoters. ( H ) Scatter plot showing differentially hypermethylated CpGs in sgIL1RN day-3 cells. Blue dots indicate significantly hypermethylated CpGs (Δβ ≥ 0.3, FDR < 0.05, n = 13). Yellow dots highlight IL1RN probes shown in (E). ( I ) MA plot showing DEGs in sgIL1RN day-3 cells. ( J ) Upset plot depicting overlap of the dCas9-DNMT3A–binding sites, hypermethylated CpGs, and DEGs. ( K ) DNAm dynamics (array data) of two significantly hypermethylated IL1RN promoter CpGs. Unpaired two-tailed Student’s t test, n = 4, means ± SEM, (**** P < 0.0001). ( L ) IL1RN dynamics (RNA-seq) in CTRL and edited cells. Unpaired two-tailed Student’s t test, n = 2, means ± SEM, (*** P < 0.001).
Article Snippet: The design of the sgRNAs targeting the IL1RN promoter (chr2:113,127,440-113,127,701) and CTRL regions was performed using Benchling sgRNAs design tool for CRISPR ( https://benchling.com/crispr ). sgRNAs close to a protospacer adjacent motif with 5′-NGG-3′ were selected with the best on-target and off-target scores.
Techniques: ChIP-qPCR, Negative Control, Two Tailed Test, Expressing, Quantitative RT-PCR, Binding Assay, RNA Sequencing
![( A ) Schematic of the dCas9-TET1 IL1RN promoter epigenome editing. The four <t>sgRNAs</t> targeting the IL1RN TSS are shown. #1 to #4 indicate the CpGs analyzed in (C). ( B ) ChIP-qPCR showing dCas9-TET1 enrichment at the IL1RN promoter. A 5-kb downstream region from the TSS is shown as a negative control region. Unpaired two-tailed Student’s t test, n = 3, mean SEM, (**** P < 0.0001). ( C ) DNAm levels (pyrosequencing) at four CpGs upstream of IL1RN TSS [as in (A)] in CTRL and edited B cells. One-way analysis of variance (ANOVA) with Dunnett’s post hoc correction, n = 3, means ± SEM, (* P < 0.05; ** P < 0.01, *** P <<0.001,**** P < 0.0001). ( D ) Top, IL1RN expression (RT-qPCR) in CTRL and edited B cells. Unpaired two-tailed Student’s t test, n = 3, means ± SEM, (*** P < 0.001). Bottom, IL1RN protein levels in CTRL and edited B cells. Fold change normalized to β-actin (CTRL versus sgIL1RN). ( E ) Schematic of the dCas9-DNMT3A IL1RN promoter epigenome editing experiment as in (A), showing two Infinium MethylationEPIC v2.0 probes. ( F ) Genome browser snapshot showing dCas9-DNMT3A binding at the IL1RN promoter. The location of the four sgRNAs targeting the IL1RN promoter and the two array probes is depicted. ( G ) Scatter plot showing differential dCas9-DNMT3A enrichment in sgIL1RN B cells. Large dots indicate top-bound promoters. ( H ) Scatter plot showing differentially hypermethylated CpGs in sgIL1RN day-3 cells. Blue dots indicate significantly hypermethylated CpGs (Δβ ≥ 0.3, FDR < 0.05, n = 13). Yellow dots highlight IL1RN probes shown in (E). ( I ) MA plot showing DEGs in sgIL1RN day-3 cells. ( J ) Upset plot depicting overlap of the dCas9-DNMT3A–binding sites, hypermethylated CpGs, and DEGs. ( K ) DNAm dynamics (array data) of two significantly hypermethylated IL1RN promoter CpGs. Unpaired two-tailed Student’s t test, n = 4, means ± SEM, (**** P < 0.0001). ( L ) IL1RN dynamics (RNA-seq) in CTRL and edited cells. Unpaired two-tailed Student’s t test, n = 2, means ± SEM, (*** P < 0.001).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6127/pmc12266127/pmc12266127__sciadv.adt1644-f3.jpg)