While even a technology with a future as bright as epigenome editing can have its cold day in the sun, the Suntag system has just cleared the clouds over a growing pain of epigenetic editing: the global off-target effects of dCas9-DNMT3A direct fusion constructs.
To cure our epigenetic editing blues, the lab of Ryan Lister at the University of Western Australia offers up some of their Aussie sunshine. Suntag is a repetitive peptide array, fused to dCas9 and recognized by effector domains fused to single-chain antibodies for the Suntag GCN4 peptide. The presence of multiple effector domains and antibody fusions leads to a localized stacking effect and a larger scale epigenetic editing event.
Needless to say, the Suntag system has been a “disruptive” technology for the field of epigenome editing, particularly when it comes to DNA (de)methylation, where we’ve seen both dCas9-Suntag-DNMT3A and dCas9-Suntag-TET1. To compare dCas9-DNMT3A to dCas9-Suntag-DNMT3A, Pflueger et al. employed targeted bisulfite sequencing (targeted bsPCR-seq) and examined select loci in HeLa (and MC4-7) cells.
Here’s what shined through when they examined the direct fusion constructs:
- At “dim” levels of expression, dCas9-DNMT3A establishes low levels of on-target methylation at the UNC5C locus (21% largest single CpG increase, 5% average increase)
- “Bright” levels of dCas9-DNMT3A expression result in on-target methylation (52% largestsingle CpG increase, 16% average increase) but also stronger off-target effects at the BCL3 promoter (3% average increase for high expression, 0.8% for low expression)
dCas9-Suntag-DNMT3A Delivers Specific and High Levels of DNA Methylation
With no optimal balance of on-target and off-target effects for the dCas9-DNMT3A direct fusion in sight, the team turned to the dCas9-Suntag-DNMT3A system and returned with blinding insight:
- First, they employed a titration series to balance on-target and off-target effects and select the best dCas9-Suntag-DNMT3A system
- The team captured the “Goldilocks zone” of on-target methylation at the UNC5C locus (49% largest single CpG increase, 12.6% average increase) and off-target DNA methylation at the BCL3 locus (only 0.7%)
- Targeting dCas9-Suntag-DNMT3A to multiple CTCF sites via different sgRNAs also illuminated Suntag’s multiplexing capabilities
- They discovered a less than 5% loss in methylating power when compared to targeting the sites on their own
The Genome-Wide Search for Off-Target Effects
The team then took a global perspective and added chromatin immunoprecipitation (ChIP) and ChIP bisulfite sequencing (ChIP-bs-seq) into the mix. To fine-tune their system, they added different fluorescent tags to dCas9-Suntag, the antibody and DNMT3A fusion, as well as a sgRNA for an intronic CTCF binding site in the SHB locus. These fluorescent markers were subjected to flow cytometry to select for cells with a medium level of expression for all components. The group found that only three of the off-target sites bound by dCas9-Suntag-DNMTA show a greater than 10% increase of methylation at a single CpG. Notably, these sites show a strong sequence match to the sgRNA, suggesting that sgRNA design represents the source of off-target effects.
Next, the team utilized Illumina’s TruSeq Methyl Capture EPIC kit for targeted bisulfite sequencing of 2.6 million CpGs to confirm their findings of limited off-target effects. Finally, they demonstrated the system’s power for basic biological insight by targeting binding sites for the methylation-sensitive transcription factors CTCF and NRF1, where they induced large changes in methylation that reduced transcription factor binding. Overall, this work demonstrates that dCas9-Suntag-DNMT3A offers a specific and tunable tool for precision DNA methylation and also leaves us eager to see the potential of Suntag with multiple epigenetic effector domains.
Shine some light on your quest for precision DNA methylation over at Genome Research, June 2018