Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a Diagram illustrating the principle of site-specific demethylation with dCas9 removal in order to facilitate transcription factor binding to the newly demethylated region. First, DNA is endogenously methylated by Dnmt1 with every round of replication and RNA polymerase II (RNA-polII) is not recruited to the promoter. After the introduction of dCas9 and a promoter-targeting gRNA, Dnmt1 is physically occluded from the locus and nascent strands of DNA are unmethylated, facilitating passive demethylation of the bound region. However, RNA-polII is also physically occluded by dCas9. If dCas9 is successfully removed, the unmethylated DNA no longer serves as a substrate for Dnmt1 and continues to remain unmethylated and RNA-polII may now be recruited. b Methylation of CpGs 9, 10, and 11 (mean ± SEM) which had been previously demethylated by high-puromycin gRNA3:dCas9 in NIH-3T3 cells, after 75 days of passaging following the lentiviral transduction of Cre recombinase (pink) or empty-vector control (red) ( n = 5 biologically independent samples). c Il33-002 expression (mean ± SEM) in NIH-3T3 cell lines stably expressing gRNAscr (gray) or gRNA1 (blue), gRNA2 (purple), or gRNA3 (pink) under high-puromycin conditions in combination with dCas9, followed by dCas9 removal by Cre recombinase as assayed by RT-qPCR and normalized to Actb expression. Statistical comparisons are to gRNAscr condition ( n = 4 biologically independent samples). d , e Il33-002 expression ( d n = 4–5 biologically independent samples) or Il33-001 expression ( e n = 5 biologically independent samples) in NIH-3T3 cells from ( c ) following treatment wither water control or 1 µM 5-aza-2′-deoxycytidine, measured by RT-qPCR and normalized to Actb expression (mean ± SEM). f Il33-002 expression (mean ± SEM) measured by RT-qPCR and normalized to Actb expression, in dCas9:gRNAscr (gray) or dCas9:gRNA3 (pink) NIH-3T3 cells following Cre recombinase treatment and then treated with poly(I:C) (1 µg/mL) or water control for 4 or 8 h ( n = 3 biologically independent experiments). g DNA methylation (mean ± SEM) assayed by bisulfite-pyrosequencing in NIH-3T3 cells expressing dCas9, gRNAscr, and Cre treated with 1 µg/mL poly(I:C) or water control for 8 and 24 h ( n = 3 biologically independent experiments). h Summary of maximal Il33-002 induction (mean ± SEM) (left y -axis, pink bars; data in log2 scale but axis numbering is not transformed) and maximal promoter demethylation (purple, right y -axis, calculated as percent unmethylated divided by control methylation) under different treatments presented thus far ( x -axis: dCas9, 5-aza-2′-deoxycytidine, dCas9-VP64, dCas9-TET, and dCas9-deadTET). Where relevant, data for maximally inducing/demethylating gRNA is shown. n = 3–6 biologically independent cell cultures * indicates statistically significant difference of P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns not significant (Student’s t -test, two-sided, with Holm-Sidak correction if number of tests is greater than 3). Source data are provided as a Source Data file.

Article Snippet: A lentiviral backbone was obtained from Addgene (pLenti-puro, Addgene #39481) and the CMV promoter was removed to prevent aberrant transcription by digesting the plasmid with ClaI-HF and BamHI-HF (NEB), gel extracting, removing DNA overhangs with the Quick BluntingTM Kit (NEB), and circularization with T4 Ligase (Thermo Fisher Scientific) for 1 h at 22 °C.

Techniques: Binding Assay, Methylation, Passaging, Transduction, Plasmid Preparation, Control, Expressing, Stable Transfection, Quantitative RT-PCR, DNA Methylation Assay, Transformation Assay

Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a Clustering of NIH-3T3 samples with indicated lentiviral treatment and replicate number by CpG methylation, based on highly covered (≥10×) CpGs common to all samples with the cluster using Samples function in the methylKit package for R (ward.D2 method). b Fraction of total sequenced CpGs (mean ± SEM) that read as methylated (after bisulfite conversion) in each treatment type, aligned and calculated with Bismark default parameters ( n = 3, biological replicates; ** P < 0.01 with two-sided Student’s t -test). c Number of significantly differentially methylated CpGs (dmCpGs) (red = hypomethylated, blue = hypermethylated) determined by methylKit calculateDiffMeth function (≥5× coverage, n = 3, q -value ( p -value adjusted for multiple testing by SLIM method) <0.01, 25% methylation difference) of dCas9TET:gRNAscr and dCas9TET:gRNA3 NIH-3T3 cell lines compared to untreated control NIH-3T3 cells (left) or compared to dCas9:gRNAscr:Cre or dCas9:gRNA3:Cre, respectively. d Genome browser view of mouse (mm10 genome) chromosome 1, with bedGraphs containing hypomethylated dmCpGs (and amount of hypomethylation in %) in dCas9-TET:gRNA3 (top, blue) and dCas9-TET:gRNAscr (middle, light blue) from ( c , right, chromosome 1 only) and dCas9:gRNA3:Cre hypomethylated dmCpGs compared to dCas9:gRNAscr (bottom, pink), which are the same as pink inset in e . Range is 0 to −100. Gene structures are densely mapped and sparsely labeled at the bottom (dark blue). e Manhattan plot of all hypomethylated (>25% change in methylation) sites in dCas9:gRNA3:Cre cells compared to dCas9:gRNAscr:Cre. Significantly differentially methylated sites were considered under default methylKit conditions ( q < 0.01, above horizontal blue line). CpGs circled in red and labeled with q-values represent two target Il33-002 CpGs (10 and 11) and the CpG circled in black represents the third-highest (top non-target) dmCpG ranked by q -value. Pink box highlights significant dmCpGs in chromosome 1, which are displayed in d . f Best alignments to gRNA3 and PAM sequence of four 100 bp regions surrounding (±50 bp) four selected off-target dmCpGs (of 641 total) from e . Dashes indicate mismatches. Vertical lines indicate matching base pairs. Matched base pairs in off-target region are also shown in blue. For the purposes of this representation, the adenine in the NAG PAM in considered a mismatch to the more active guanine in the NGG PAM. Top alignment (in black) shows the region containing the top off-target dmCpG by q -value, chr8:4802686, circled in black in e (14/23 mismatches). Second from top alignment displays the 100 bp off-target region containing a sequence with the most similarity to gRNA3 of all 641 100 bp off-target regions, as calculated by position-weighted mismatch algorithm CCTop (ten mismatches). Third from top alignment displays off-target region ranked second for similarity to gRNA3 by CCTop (eight mismatches but one is closer to 3′/PAM end than above). Final alignment shows the off-target region with the lowest mismatches overall to gRNA3, regardless of position, which is 7. g Volcano plot of ChIP-seq significantly differentially enriched regions in gRNA3 and gRNAscr conditions ( n = 3) for anti-FLAG ChIP-seq against FLAG-dCas9, using input as control. Log2 fold change (log2(gRNAscr) − log2(gRNA3)) is plotted on the x -axis and −log10(False Discovery Rate) is plotted on the y -axis. The locus corresponding to the Il33-002 transcription start site is circled in red. h Genome browser view (mm10) of Il33-002 (blue). Statistically significant peak (turquoise) (circled in red in g ), peak summit (purple) and gRNA3 sequence (pink) are labeled. i Manually curated sequence alignments of top five DERs from ChIP-seq data to gRNA3 and PAM sequence. Identical sequence matches are marked in blue and bolded. Potential gaps are indicated with dashes. Chromosomal locations are given for the mm10 genome build. j Volcano plot depicting changes in methylation and associated statistical probabilities in dCas9:gRNA3 NIH-3T3 cells (from WGBS data) for CpGs that are within 150 differentially enriched off-target regions bound by dCas9:gRNA3 ( Il33-002 is excluded). Change in methylation ( x -axis) is expressed as mean percent methylation in dCas9:gRNAscr subtracted from mean percent methylation in dCas9:gRNA3. Statistical probabilities are provided as the −log10 of the p-value derived by the independent t -test as corrected for multiple testing by the False Discovery Rate method. Five hundred and forty-nine CpGs were located in DERs after filtering for CpGs that were 5× covered in all six samples; however, 132 CpGs with exactly 0% methylation in all six samples and 13 CpGs with exactly 100% methylation in all six samples are not depicted as P -values cannot be mathematically calculated. Therefore, 404 CpGs are shown. Underlying WGBS data was presented above and reflects n = 3 independent stable cell lines. Source data are provided as a Source Data file.

Article Snippet: A lentiviral backbone was obtained from Addgene (pLenti-puro, Addgene #39481) and the CMV promoter was removed to prevent aberrant transcription by digesting the plasmid with ClaI-HF and BamHI-HF (NEB), gel extracting, removing DNA overhangs with the Quick BluntingTM Kit (NEB), and circularization with T4 Ligase (Thermo Fisher Scientific) for 1 h at 22 °C.

Techniques: CpG Methylation Assay, Methylation, Control, Labeling, Sequencing, ChIP-sequencing, Derivative Assay, Stable Transfection

Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a The sequence of the lentiviral gRNAHNF4A and its PAM site in blue. Above is the reference sequence of the HNF4A gene near the gRNA target site, as validated by Sanger sequencing in primary human hepatocytes expressing via lentivirus Cas9 and gRNAscr, with the TSS indicated by a black arrow and the reference protein sequence in red. CGs are bolded and underlined. Below is the dominant Sanger sequence profile of a primary human hepatocyte population expressing lentiviral Cas9 and gRNAHNF4A. This mutation and the resulting difference in the amino acid sequence, as well as the reference sequences at this location, are highlighted in yellow. b Two technical replicates each of the Sanger sequencing chromatograms from the primary human hepatocytes expressing dCas9 and gRNAscr (left) or dCas9 and gRNAHNF4A (right) at the targeted HNF4A locus. c Sanger sequencing results of 13 gRNAscr and 12 gRNAHNF4A DNA strands following bisulfite conversion from the cell populations in ( b ), demonstrating both the methylation levels and the variety of mutations induced by Cas9 in gHNF4A-treated cells. d Same as ( c ) except data expanded is expanded to a larger (>300 bp) region, and simplified such that only CpGs are shown, where blue squares indicate unmethylated CpGs, red squares indicate methylated CpGs, and white squares indicate missing information due to Cas9-induced deletions. CpGs are numbered in accordance with ( a ). e Bisulfite-sequencing data from ( d ) (center) as well as five CpGs immediately upstream (left) and seven CpGs immediately downstream (right), displayed as percent DNA methylation over all sequenced DNA strands in primary human hepatocytes expressing Cas9 and either gRNAscr (gray) or gRNAHNF4A (orange) and as mean ± SD as it is summary data from one mutated cell line. Individual dots represent individual strands of DNA from this clonal cell line. f HNF4A expression in primary human hepatocytes expressing Cas9 and either gRNAscr (gray) or gRNAHNF4A (orange) quantified by RT-qPCR and normalized to GAPDH expression, followed by normalization to average expression in gRNAscr cells, with a dashed line at 1 ( n = 6 independent clones, mean ± SD). * indicates statistically significant difference of P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns not significant (Student’s t -test, two-sided, with Holm-Sidak correction if number of tests is greater than 3). Source data are provided as a Source Data file.

Article Snippet: A lentiviral backbone was obtained from Addgene (pLenti-puro, Addgene #39481) and the CMV promoter was removed to prevent aberrant transcription by digesting the plasmid with ClaI-HF and BamHI-HF (NEB), gel extracting, removing DNA overhangs with the Quick BluntingTM Kit (NEB), and circularization with T4 Ligase (Thermo Fisher Scientific) for 1 h at 22 °C.

Techniques: Sequencing, Expressing, Mutagenesis, Methylation, Methylation Sequencing, DNA Methylation Assay, Quantitative RT-PCR, Clone Assay