Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) The constructs showing the dead Cas9 (dCas9) fused with p300core HAT domain (dCas9-p300 C ) and expressed from cytomegalovirus (CMV) promoter. The small guide RNA (sgRNA) designed for hs1.2 and hs4 enhancers were expressed from the U6 promoter. The cotransfection of dCas9-p300 C and either sgRNAs (hs1.2/hs4) in CH12-F3-2A cells showing site-specific enhancer activation at 3′RR. ( B ) The ChIP-qPCR showing the enrichment of H3K27ac histone in dual-transfected dCas9-p300 C with either sgRNAs in Med12 KD cells. ( C ) Corresponding RT-qPCR data showing the enhancer transcripts level. ( D ) The bar plot showing the IgA rescue efficiency by dCas9-p300 C –mediated enhancer activation in Med12 KD CH12F3-2A cells. The black bar and gray bar showing the control samples. The left blue bars showing the rescue efficiency in WT R cells. The right two blue bars showing the dual-transfected dCas9-p300 C with either sgRNAs (hs1.2 and hs4) in Med12 KD cells. ( E ) LM-PCR assay estimating DNA break rescue in indicated samples as described above. Right: The ImageJ analysis showing the band intensities in the respective samples. ( F and G ) The ChIP assay was performed using the indicated antibodies, followed by qPCR showing the enrichment at S regions in dual-transfected dCas9-p300 C with sgRNAs (hs1.2 and hs4) in Med12 KD cells. ( H ) The 3C-PCR assay showing long-range interaction (LRI) in dual-transfected dCas9-p300 C with Med12 KD cells. Bottom: The ImageJ analysis showing the band intensities in the respective samples. ( I ) The ChIP-qPCR showing the AID enrichment in dual-transfected dCas9-p300 C with sgRNAs in Med12 KD cells. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05).

Article Snippet: The vector dCas9-KRAB-MeCP2 was purchased from Addgene (no. 11082).

Techniques: Construct, Cotransfection, Activation Assay, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A and B ) The ChIP-qPCR showing the occupancy of Jmjd6 at 3′RR in control and either Jmjd6- or Med12-depleted cells. ( C ) Top: The FACS data showing the effect of Jmjd6 and Carm1 KD by respective siRNAs. Bottom left: The RT-qPCR data showing the KD efficiency by respective samples, and the data were normalized with β2m abundance. ( D ) The bar plot showing the effect of Jmjd6 and Carm1 KD on 3′RR transcripts. ( E and F ) The ChIP assay was performed by indicated the antibodies, followed by qPCR in control and Jmjd6 KD cells. ( G ) The bar plot showing the IgA rescue efficiency in dual-transfected dCas9-p300 C with hs4 sgRNAs in Jmjd6 and/or Carm1 KD cells. ( H ) The FACS analysis showing the IgA rescue efficiency in methylation-defective Med12 mutants in Med12 KD cells. The position of the mutations at PQL domain is shown at the right. ( I ) The RT-qPCR analysis showing the rescue of hs1.2 and hs4 transcripts in WT R and ΔPQL Med12 mutant in Med12 KD cells. ( J ) The schematic representation showing the sequential steps of Med12 workflow in CSR. Med12 is methylated by Jmjd6/Carm1 complex (magenta) at different positions (black boll). Methylation recruits p300 protein to 3′RR, which marks histone H3K27 acetylation and activate the enhancers. Activated enhancers were transcribed into eRNA, which regulates H3K4me3 at S region and recruits DNA break and repair complex for CSR. The result summarizes the means ± SD of three independents experiments and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05). NHEJ, nonhomologous end joining.

Article Snippet: The vector dCas9-KRAB-MeCP2 was purchased from Addgene (no. 11082).

Techniques: Quantitative RT-PCR, Transfection, Methylation, Mutagenesis, Two Tailed Test

Journal: Genes

Article Title: Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.

doi: 10.3390/genes14040906

Figure Lengend Snippet: Figure 1. Establishment of CRISPRa and CRISPRi expressing cell lines (A) Schematic representation of CRISPRa vectors. CMV, cytomegalovirus promoter; Cas9m4, inactive Cas9 lacking endonuclease activity; VP, activation domain VP64; Rta, Epstein–Barr virus R transactivator; p300 Core, p300 histone acetyltransferase (B) RT-PCR analysis in the established CRISPRa DF-1 cell lines. (C) Schematic representation of CRISPRi vectors. dCas9, inactive Cas9 lacking endonuclease activity; K, Krüppel- associated box, KRAB; MeCP2, methyl-CpG binding protein 2 (D) RT-PCR analysis in the established CRISPRi DF-1 cell lines. The endogenous GAPDH gene was used as a control.

Article Snippet: To generate the CRISPRi cell line, 2.5 × 105 DF-1 cells were transfected with 200 ng of piggyBac transposon vectors containing CRISPRi components (pB-CAGGS-dCas9, pBCAGGS-dCas9-KRAB or pB-CAGGS-dCas9-KRAB-MeCP2) which were gifts from Alejandro Chavez and George Church (Addgene plasmid #110823, #110822, and #110824, respectively) and 50 ng of transposase vector (PB200, Systems bioscience, Palo Alto, CA, USA) using the Lipofectamine 3000 (Thermo Fisher–Scientific) according to the manufacturer’s instructions [21].

Techniques: Expressing, Activity Assay, Activation Assay, Virus, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Control

Journal: Genes

Article Title: Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.

doi: 10.3390/genes14040906

Figure Lengend Snippet: Figure 2. The regulation of the chicken HMGA1, SMARCB1, IRF7 and PPARG gene expression in CRISPRa and CRISPRi DF-1 cells. Gene structure and quantitative analysis of HMGA1 (A), SMARCB1 (B), IRF7 (C) and PPARG (D) in the gRNAs-treated CRISPRa and CRISPRi cell lines. The targeting positions of the gRNAs for each gene (1, 2 and 3 or 1, 2, 3, 4 and 5) were marked. Grey rectangles indicate exons. Scale bars, 1 Kb. Data are shown as the mean ± SEM. Significant differences between the mock controls and gRNAs-transfected samples were determined by Student’s t-test. Statistical significance was marked as * p < 0.05, ** p < 0.01.

Article Snippet: To generate the CRISPRi cell line, 2.5 × 105 DF-1 cells were transfected with 200 ng of piggyBac transposon vectors containing CRISPRi components (pB-CAGGS-dCas9, pBCAGGS-dCas9-KRAB or pB-CAGGS-dCas9-KRAB-MeCP2) which were gifts from Alejandro Chavez and George Church (Addgene plasmid #110823, #110822, and #110824, respectively) and 50 ng of transposase vector (PB200, Systems bioscience, Palo Alto, CA, USA) using the Lipofectamine 3000 (Thermo Fisher–Scientific) according to the manufacturer’s instructions [21].

Techniques: Gene Expression, Transfection

Journal: Genes

Article Title: Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.

doi: 10.3390/genes14040906

Figure Lengend Snippet: Figure 3. The regulation of the chicken SMARCB1 gene expression by individual gRNAs in CRISPRa and CRISPRi DF-1 cells. (A) Gene structure of SMARCB1 gene and the location of gRNAs (1, 2 and 3).

Article Snippet: To generate the CRISPRi cell line, 2.5 × 105 DF-1 cells were transfected with 200 ng of piggyBac transposon vectors containing CRISPRi components (pB-CAGGS-dCas9, pBCAGGS-dCas9-KRAB or pB-CAGGS-dCas9-KRAB-MeCP2) which were gifts from Alejandro Chavez and George Church (Addgene plasmid #110823, #110822, and #110824, respectively) and 50 ng of transposase vector (PB200, Systems bioscience, Palo Alto, CA, USA) using the Lipofectamine 3000 (Thermo Fisher–Scientific) according to the manufacturer’s instructions [21].

Techniques: Gene Expression

Journal: Genes

Article Title: Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit.

doi: 10.3390/genes14040906

Figure Lengend Snippet: Figure 4. The regulation of the chicken IRF7 gene expression by individual gRNAs in CRISPRa and CRISPRi DF-1 cells. (A) Gene structure of IRF7 gene and the location of gRNAs (1, 2, 3, 4 and 5). The

Article Snippet: To generate the CRISPRi cell line, 2.5 × 105 DF-1 cells were transfected with 200 ng of piggyBac transposon vectors containing CRISPRi components (pB-CAGGS-dCas9, pBCAGGS-dCas9-KRAB or pB-CAGGS-dCas9-KRAB-MeCP2) which were gifts from Alejandro Chavez and George Church (Addgene plasmid #110823, #110822, and #110824, respectively) and 50 ng of transposase vector (PB200, Systems bioscience, Palo Alto, CA, USA) using the Lipofectamine 3000 (Thermo Fisher–Scientific) according to the manufacturer’s instructions [21].

Techniques: Gene Expression

Journal: Viruses

Article Title: Validation of Candidate Host Cell Entry Factors for Bovine Herpes Virus Type-1 Based on a Genome-Wide CRISPR Knockout Screen

doi: 10.3390/v16020297

Figure Lengend Snippet: CRISPRi mediates efficient KD of gene expression in MDBK cells. ( a ). Schematic representation of the CRISPRi system applied in this study, based on the module developed by Andrea Califano et al. (See Materials and Methods). ( b ). Doxycycline inducible expression of the dCas9-KRAB-MeCP2 module from a stably integrated expression cassette at the cow rosa26 locus. Green heptagram represents doxycycline, purple squares are rTTA expressed from the downstream expression cassette, used to enhance doxycycline-driven induction. ( c ). Targeting strategy to knock-in the dCas9-KRAB-MeCP2 cassette into rosa26 using TAL1.6 stimulated HDR) and Hygromycin selection, prior to dilutional cloning and genotyping by PCR using primer set F + R (red arrows) binding outside the homology arms. wt: wild type; tg: targeted. ( d ). Representative genotyping results by PCR from a homozygous targeted clone (+/+), and two heterozygotes (+/wt), black arrows point to amplicons from the targeted allele (top, 12 kb PCR product) and wt allele (bottom, 4 kb PCR product). ( e ). Knockdown efficiency of Doxycycline induced CRISPRi under six Doxycycline concentrations across five genes represented as mRNA level measured by reverse transcription and qPCR at 48 h post induction. ( f ). Relative expression levels of five genes in dCas9+/+ MDBKs each targeted by three CRISPRi guides (g1, g2, g3) compared to cells transfected with non-targeting CRISPRs (NC). These results are based on RT-qPCR results from a single experiment with three technical qPCR repeats, error bars represent +/- 1 standard error of the mean.

Article Snippet: The dCas9 targeting vector was created by Gibson Assembly, incorporating the inducible dCas9-KRAB-MeCP2 fusion cassette from PB-TRE-dCas9-KRAB-MeCP2 (gift from Andrea Califano, Addgene # 122267), the EF1a promoter driven rtTA fragment from PB-CA-rtTA Adv (gift from Andras Nagy, Addgene # 20910), and an in-frame Hygromycin selection marker connected by 2A peptide sequence.

Techniques: Expressing, Stable Transfection, Knock-In, Selection, Clone Assay, Binding Assay, Transfection, Quantitative RT-PCR