Method overview To achieve targeted locus and allele-specific DNA demethylation, HEK293 cells were transfected with two plasmids. One plasmid contains, dCas9 fused to a SunTag with five repeats of the GCN4 peptide, separated by 22 aa long linkers, and scFv-fused TET1CD, as well as a GFP reporter protein. The other plasmid is a multiguide plasmid with 4 individual sgRNAs flanked by U6 promoter and gRNA scaffold, and a DsRed fluorophore. Control experiments were conducted with a scrambled sgRNA that does not have a binding site in the human genome. Initial studies showed that cells positive for two plasmids exhibited detectable fluorescence of the corresponding reporter proteins on day 3 post-transfection. Hence, FACS sorting was conducted at this time point. A part of the sorted cells was used immediately for downstream analysis, the other part was re-seeded to harvest at later time points. For DNA methylation analysis, genomic DNA was isolated from the cell samples and subjected to bisulfite treatment. Library preparation was performed using the bisulfite-converted samples, followed by NGS and data analysis. All methylation experiments were conducted in three independent biological replicates. For measurement of the genomic allele frequencies, genomic DNA of the untreated samples was used for the amplification of the region around the target SNP and an exonic region with additional SNP for each target, which was followed by library preparation, NGS and data analysis. To monitor the variation in the expression of the target genes, RNA was isolated from the treated cells on Day 6. cDNA synthesized from the isolated RNA was used for the library preparation of the exonic region. The library was subjected to NGS followed by data analysis. All experiments were conducted in three independent biological replicates. Method details The gDNA of transfected HEK293 cells sorted by FACS was extracted using QIAmp DNA Mini Kit (Qiagen). 500 ng of genomic DNA was subjected to overnight digestion with EcoRV which is not cutting in any of the target amplicons. Zymo EZ DNA Methylation-Lightning Kit (D5030-E) was used for bisulfite conversion. The library for NGS was prepared by two consecutive PCR reactions (Leitao et al, 2018). Firstly, bisulfite converted genomic DNA of each sample was amplified with target gene specific primers. The gene specific optimized amount of a product from the first PCR was used as a template for the second PCR to add the Illumina TruSeq sequencing adapters. Final products were quantified, pooled in equimolar amounts and purified using SPRIselect beads (Beckman Coulter). Ready-to-use pools of libraries were sequenced on NovaSeq 6000 using a PE250 flow cell (Novogene). For expression analysis, RNA was isolated from the sorted cells using Qiagen RNeasy extraction kit (Cat. No. 74034). By an additional treatment with TURBO DNA-free™ Kit (Ambion #AM1907) the residual genomic DNA from the samples were removed. 500 ng of the DNase-free RNA was used for cDNA synthesis with Applied Biosystems- High-Capacity cDNA Reverse Transcription Kit (Cat No 4368814). NRT was used as a negative control for cDNA synthesis, where the reaction was conducted without addition of the reverse transcriptase enzyme. In addition, NTC (no template control) reactions were included. The transcripts were subjected to library preparation in a two-step PCR process as mentioned above. For amplification of the genomic regions, 10 ng of the isolated genomic DNA was used. Two-step library preparation was carried out for NGS of genomic regions. All NGS data were obtained in the form of FASTQ files. Data analysis NGS data in a FASTQ format was analyzed as described (Rajaram et al., 2023) on the Galaxy platform (https://usegalaxy.org/) (The Galaxy platform for accessible, reproducible and collaborative biomedical analyses, 2022), where all the following tools are available. First, Illumina adapter sequences were removed using Trim Galore!. Afterwards, two paired-end reads were merged using Pear and reads with low quality were removed with Filter FASTQ. All NGS data files were subjected to this processing. For quantitative analysis of the methylation at individual CpG sites, the following steps were carried out. De-multiplexing of individual samples tagged with combinations of barcodes and Illumina indices was done by converting the FASTQ files using FASTQ to Tabular, followed by selection of lines with the tool Select and re-conversion of the files to a FASTQ format with Tabular to FASTQ. For the alignment of reads to a reference sequence, bwameth was used and the DNA methylation at each CpG site was analyzed by applying the tool MethylDackel. The output files were processed using Microsoft Excel. For the analysis of the allelic ratios of the transcript and genomic region, de-multiplexing of individual samples tagged with combinations of barcodes and Illumina indices was done by converting the FASTQ files using FASTQ to Tabular, followed by selection of lines with the tool Select. Input for the selection of lines was provided in accordance to the SNP of interest. Output of the tool Select provides the number of reads corresponding to each allele.

References The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic acids research 2022, 50, W345-W351, doi: 10.1093/nar/gkac247 Rajaram, N.; Kouroukli, A.G.; Bens, S.; Bashtrykov, P.; Jeltsch, A. Development of super-specific epigenome editing by targeted allele-specific DNA methylation. Epigenetics Chromatin 2023, 16, 41, doi: 10.1186/s13072-023-00515-5