Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a , Schematic representation of characterization of CHD ncDNVs in iPSC-CMs. CRISPR–Cas9 was used to introduce ncDNVs from CHD lentiMPRA into their endogenous loci. After isolation of clonal lines and differentiation to iPSC-CMs, the expression of neighboring genes was measured by RT–qPCR. b – e , Validated ncDNVs and their impact on neighboring CHD-associated genes. Bar plots show RT–qPCR analysis of genes adjacent to DNVs near BCOR ( b ), MYOCD ( c ), ADAMTS6 ( d ), and ACVRL1 ( e ) in day 17 iPSC-CMs. Data are shown as mean ± s.d. of at least three independent experiments. ANOVA with Dunnett’s test compared to wild-type (WT) control. REF and ALT sequences are shown to the right, with the SNV highlighted in yellow, along with predicted transcription factor binding motifs impacted by SNVs. BCOR knock-in lines, n = 3. All others, n = 4. f , g , UMAP projection of wild-type and ncDNV knock-in nuclei from iPSC-CMs at day 10 of differentiation. f , iPSC-CM clusters are colored and numbered 0–8. g , Nuclei are colored by genotype. Merge (top-left) shows all genotypes with indicated colors and the remaining panels each show one genotype. h , Stacked bar graph of the percentages of nuclei in each cluster. The proportion of nuclei in each cluster was compared to wild-type nuclei; numbers indicate significant P values (one-way ANOVA with Dunnett’s multiple comparison test). i , Heatmap of genes that were differentially expressed in BCOR or MYOCD ncDNV knock-ins compared to wild type. Genes that were significantly different from wild-type in both replicates (Seurat FindMarkers P adj < 0.05; ) were selected. Heatmap displays the scaled average gene expression from each replicate. j , k . Gene Ontology analysis of the genes differentially expressed in both MYOCD ncDNV homozygous and heterozygous lines ( j ) or in both BCOR ncDNV lines ( k ) compared to wild-type iPSC-CMs. Hypergeometric test with Bonferroni correction for multiple testing. In b – e , **** P < 0.0001.

Article Snippet: To introduce CHD ncDNVs into iPS cells, we used WTC-11 cells in which dox-inducible Cas9 (ref. ) is inserted into the AAVS1 locus (WTC-Cas9 iPS cell line). sgRNAs targeting regions near the ncDNVs of interest were designed using CHOPCHOP and transcribed in vitro using the EnGen sgRNA Synthesis Kit (NEB, E3322S).

Techniques: CRISPR, Introduce, Isolation, Expressing, Quantitative RT-PCR, Control, Binding Assay, Knock-In, Comparison

Journal: Hepatology Communications

Article Title: Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

doi: 10.1002/hep4.2014

Figure Lengend Snippet: Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 targets covalently closed circular DNA (cccDNA) and exerts an antiviral effect accompanied by a significant reduction in cccDNA in HBV‐infected hepatoma cells. (A) Western blot of Cas9 and b‐actin in HepG2‐hNTCP‐C4‐iCas9 cells 1 day after doxycycline (DOX) treatment. (B) Representative images of immunofluorescent staining of hepatitis B core (HBc) 13 days after hepatitis B virus (HBV) inoculation. (C–F) HepG2‐hNTCP‐C4‐iCas9 cells lentivirally transduced with the HBV guide RNA (gRNA) or NC gRNA vector were inoculated with HBV (10,000 GEq/cell). (C) Experimental protocol. (D) Intracellular cccDNA levels in HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (10,000 GEq/cell) (n = 4, ** p < 0.01). (E) Indel ratio in the cccDNA of HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (n = 4, * p < 0.05). (F) Intracellular pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels in HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (10,000 GEq/cell) (n = 4, ** p < 0.01). DAPI, 4′,6‐diamidino‐2‐phenylindole. NC, negative control.

Article Snippet: HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells, [ ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.

Techniques: CRISPR, Infection, Western Blot, Staining, Virus, Transduction, Plasmid Preparation, Negative Control

Journal: Hepatology Communications

Article Title: Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

doi: 10.1002/hep4.2014

Figure Lengend Snippet: HBV‐CRISPR exerts an antiviral effect accompanied by a significant reduction in cccDNA in HBV‐infected primary human hepatocytes (PHHs). PHHs isolated from humanized liver chimeric mice were lentivirally transduced with tandem Cas9 gRNA and HBV gRNA (or NC gRNA)–expressing vectors (10 multiplicity of infection [MOI]) 19 days after HBV inoculation (500 GEq/cell). (A) Experimental protocol. (B) cccDNA levels, intracellular pgRNA levels, and supernatant HBV DNA, HBs antigen, and HBe antigen levels in PHHs 35 days after HBV inoculation (n = 4; * p < 0.05, ** p < 0.01).

Article Snippet: HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells, [ ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.

Techniques: CRISPR, Infection, Isolation, Transduction, Expressing

Journal: Hepatology Communications

Article Title: Inhibition of nonhomologous end joining‐mediated DNA repair enhances anti‐HBV CRISPR therapy

doi: 10.1002/hep4.2014

Figure Lengend Snippet: Olaparib enhances the antiviral effect of HBV‐CRISPR in HBV‐infected hepatoma cells and PHHs. (A–C) HepG2‐hNTCP‐C4‐iCas9 cells transduced with HBV gRNA were treated with or without olaparib (1 μM) 10 days after HBV inoculation (10,000 GEq/cell). (A) Experimental protocol. (B) Intracellular cccDNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation. (C) pgRNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation. (D–F) HepG2‐hNTCP‐C4‐iCas9 cells transduced with HBV gRNA were treated with DOX 4 days after HBV inoculation (10,000 GEq/cell) and then with olaparib (1 μM) or vehicle 10 days after HBV inoculation. (D) Experimental protocol. (E) cccDNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (n = 4; * p < 0.05). (F) Intracellular pgRNA levels in HBV gRNA–transduced HepG2‐hNTCP‐C4‐iCas9 cells 13 days after HBV inoculation (n = 4; ** p < 0.01). (G–I) PHHs isolated from humanized liver chimeric mice were lentivirally transduced with tandem Cas9‐expressing and HBV gRNA–expressing vectors (10 MOI) 19 days after HBV inoculation (500 GEq/cell) and were then treated with olaparib (1 μM) or vehicle. (G) Experimental protocol. (H) cccDNA levels in PHHs 35 days after HBV inoculation (n = 4; * p < 0.05). (I) Intracellular pgRNA levels in PHHs 35 days after HBV inoculation (n = 4, * p < 0.05).

Article Snippet: HepG2‐hNTCP‐C4‐iCas9 cells were established through the transduction of a lentiviral vector expressing DOX‐inducible Cas9 (Lenti‐iCas9‐neo; Addgene #85400) into HepG2‐hNTCP‐C4 cells, [ ] followed by green fluorescent protein selection with fluorescence‐assisted cell sorting.

Techniques: CRISPR, Infection, Transduction, Isolation, Expressing