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Fig. 2 | BMC Biology

Fig. 2

From: Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

Fig. 2

Methylation of the HPRT1 CpG island by CRISPR-mediated sequence replacement results in HPRT1 silencing. a Percentages of Illumina sequencing reads assigned to methylated and unmethylated inserted alleles by SNVs, grouped by selection status (Pre, pre-selection; Mock, mock selection; 6-TG, 6-TG selection). Although both are enriched, methylated inserted alleles are more enriched than unmethylated inserted alleles after 6-TG selection. Wild-type sequences are not shown, but are included in percentages. The first panel shows the experiment where allele 1 was methylated and allele 2 unmethylated; the second panel shows the reciprocal experiment. Error bars show the range of triplicates. b Percentages of PacBio sequencing reads assigned to “exact matching” methylated and unmethylated inserted alleles by SNVs, grouped by selection status (Pre, pre-selection; Mock, mock selection; 6-TG, 6-TG selection). Methylated inserted alleles, but not unmethylated inserted alleles, are strongly enriched by selection. Sequences were only counted if they were in the forward orientation and exactly matched the promoter, exon 1, splice donor, PAM mutation, and one of three sets of allele-defining SNVs (wild-type, allele 1, or allele 2). Wild-type sequences are not shown, but are included in percentages. Error bars show the range of triplicates. Note that the y-axis is gapped and contains two scales, to increase resolution in 0–10% range. c Percentages of PacBio sequencing reads assigned to reverse/inverted orientation, grouped by selection status. Deletion events, as well as sequences not meeting the “exact matching” criteria defined above, were not counted. Forward-oriented sequences are not shown, but are included in percentages. The clear pattern is that inverted sequences predominate after 6-TG selection. d Observed number of methylated sites upon bisulfite sequencing of methylated, unmethylated, or wild-type alleles of the CpG island, summed across selection conditions. The region contains 35 CpG dinucleotides. Reads are assigned to in vitro methylated or unmethylated alleles, or to unedited wild-type sequence based on synonymous SNVs. In vitro methylated alleles remain heavily methylated, while unmethylated alleles and unedited sequences remain predominantly unmethylated

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