LEMONmethyl-seq tracks time-dependent changes after CRISPR epigenome editing with targeted long-read DNA methylation analysis
Background: Reading methylation across long DNA regions
The DNA methylation assay LEMONmethyl-seq captured the formation and spread of methylation around target sites after CRISPR epigenome editing in cultured cells. The study, posted as a preprint on bioRxiv, presents a method for analyzing selected DNA regions at single-base resolution and also examines methylation patterns associated with drug sensitivity in glioblastoma cell lines.
DNA methylation contributes to cell differentiation and gene expression, and it also changes during disease and aging. Conventional targeted bisulfite analysis damages DNA during processing and is generally suited to amplifying short regions of 200–300 base pairs. Reading longer regions requires a procedure that records methylation information in the sequence while limiting DNA damage. Amplifying native DNA directly by polymerase chain reaction (PCR) would otherwise erase the methylation information.
Key findings: Methylation patterns recorded by enzymatic conversion
LEMONmethyl-seq stands for Locus-Enriched Mapping Of Nucleotide methylation. The researchers used enzymatic methyl conversion (EM conversion) to protect modified cytosines and convert unmodified cytosines to uracil, then amplified the targets with the KeyPo SE polymerase. They read the sequences with Oxford Nanopore long-read sequencing, distinguishing positions that remained C from those converted to T. Because this conversion protects both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), the assay does not distinguish between the two modifications.
At the HIST2H2BE promoter, the researchers amplified about 4.5 kilobases (kb) in one piece and obtained more than 3,000 reads. The bisulfite-PCR data used for comparison comprised 7–10 Sanger reads per region, and the two methods showed similar methylation trends. The researchers reported a lower total cost for LEMONmethyl-seq. Read depth at the CD55 target was more than 300 times that of whole-genome enzymatic methyl sequencing (WGEM-seq), which was performed at an average genome-wide depth of 36-fold. This comparison is limited to the selected target and experimental conditions.
At the CD55 and CLTA promoters in HEK293T cells treated with CRISPRoff, methylation appeared at some CpG sites after 8 hours. By 48 hours, it had reached high levels across the amplified regions and remained so at 72 hours. The initial changes began around the guide RNA binding sites and then extended across broader regions.
In an experiment transitioning H9 human pluripotent stem cells from a primed to a naive state, the researchers observed reduced methylation of transposable elements. Separately, they detected CpH methylation at the CD81 target in induced pluripotent stem cell-derived neurons treated with CRISPRoff. CpH refers to a sequence in which A, C, or T follows cytosine. At 19 CpH sites in the treated group, methylation was observed in more than 3% of reads at each site.
Implications and outlook: Potential and limits in biomarker exploration
Across five glioblastoma cell lines, the researchers analyzed about 1.6 kb spanning the MGMT promoter and the start of its coding region. The two cell lines sensitive to temozolomide (TMZ) had higher average methylation than the three resistant lines. The researchers compared 120 CpG sites and found differences even outside the regions covered by existing clinical tests. This is an association among cell lines; the study did not validate performance in predicting treatment response in patients.
The analysis is limited by amplicon length and depends on an accurate reference sequence. The current procedure provides methylation information from only one DNA strand. Observations of changes around selected targets therefore cannot be extended to genome-wide off-target surveillance or clinical safety validation. The findings should be interpreted as a cell-based methodological study at the preprint stage.
Source: Full LEMONmethyl-seq study.
BACKGROUND: DNA methylation is the most prevalent epigenetic modification in human cells and undergoes dynamic changes during cell differentiation, disease progression, and aging. Here, we introduce L ocus- E nriched M apping O f N ucleotide methylation (LEMONmethyl-seq): an optimized, cost-effective pipeline for single-nucleotide detection of DNA methylation using locus-specific amplification and long-read DNA sequencing. RESULTS: We apply LEMONmethyl-seq to profile DNA methylation of endogenous gene promoters across different cell types along with DNA methylation establishment and long-range propagation induced by CRISPR epigenome editing technologies. We profile dynamic changes in DNA methylation patterns on transposable element genomic loci during global epigenetic resetting in stem cells, and we identify site-specific enrichment of non-canonical CpH methylation on genomic sites in stem cells and cultured neurons. Lastly, we apply LEMONmethyl-seq to profile DNA methylation across the CONCLUSIONS: Together, LEMONmethyl-seq serves as a cost-effective, long-read DNA methylation sequencing pipeline that advances methods for detecting DNA methylation patterns and dynamics in mammalian cells. We envision its broad use for studying chromatin pathways, diagnostics, and therapeutic applications.
Researchers studying epigenome editing can use LEMONmethyl-seq to compare when methylation forms at selected promoters and how far it spreads. For example, analyzing CD55 and CLTA at several time points after CRISPRoff treatment could help investigate how suppression of protein expression relates to the formation of DNA methylation. At MGMT, CpGs outside existing test regions could be explored as candidates for follow-up biomarker studies. Targeted analysis alone cannot establish genome-wide safety, and applying the association with drug sensitivity observed in cell lines to patients would require separate clinical validation. The reported cost advantage should also be assessed by distinguishing the comparison conditions in the study from actual testing settings.