Person:

Aryee, Martin

The biological roles of DNA methylation have been elucidated by profiling methods based on whole-genome or reduced-representation bisulfite sequencing, but these approaches do not efficiently survey the vast numbers of noncoding regulatory elements in mammalian genomes. Here we present a novel extended representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Our design strikes a balance between expanding coverage of regulatory elements and reproducibly enriching informative CpG dinucleotides in promoters, enhancers, and CTCF binding sites. Barcoded DNA fragments are pooled prior to bisulfite conversion, allowing multiplex processing and technical consistency in low input samples. Application of XRBS to single leukemia cells enabled us to evaluate genetic copy-number variations and methylation variability across individual cells. Our analysis highlights heterochromatic H3K9me3 regions as having the highest cell-to-cell variability in their methylation, likely reflecting inherent epigenetic instability of these late replicating regions, compounded by differences in cell cycle stages among sampled cells.

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1,2,3,4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.