TDAC-seq³ applies the same core concept (long-read, single-molecule deamination footprinting) to pooled CRISPR screening. Using different dsDNA deaminases (DddA11 and MGYFPDa829), TDAC-seq reads out a genetic perturbation and the surrounding chromatin structure from the same single DNA fiber, directly linking a CRISPR edit to changes in DNA accessibility on the same molecule. Those changes in DNA accessibility, such as shifts in nucleosome positioning or transcription factor occupancy, link CRISPR edits to their consequences in a way that sequencing the edit and profiling chromatin separately never could. Capturing the edit and the functional consequence simultaneously makes this approach ideal for pooled screens to evaluate how sequence motifs control accessibility of regulatory elements or the impact of single guide RNAs (sgRNAs) in DNA editing strategies.

Together, these two methods illustrate the value deaminases are bringing to single-molecule genomics: each long-read provides a detailed picture of what’s bound and accessible across kilobases of DNA, instead of capturing a population average. These methods allow researchers to understand co-occupancy, complex TF binding arrangements, and pooled genetic-perturbation outcomes all from individual reads. A lab can now capture a promoter, its distal enhancers, and the transcription factors connecting them in a single experiment, rather than piecing together the mechanistic story from several bulk experiments.

Short-read Deaminase Assays

Short-read deaminase methods build on the ATAC-seq workflow, giving up the single-molecule, multi-kilobase context of long-read sequencing in exchange for compatibility with widely available short-read instruments, such as Illumina platforms. The short-read deaminase assays are relatively simple to perform, but they require custom analysis pipelines and quality control reports to track both deamination and tagmentation. Because these methods are combined with ATAC-seq, the reads enrich for accessible regions of the genome, which are typically promoters and enhancers. Compared to long-read sequencing, these short-read sequencing deaminase assays offer high resolution footprinting in a cost-effective manner that is more widely accessible. In practice, this means labs already running ATAC-seq or short-read whole-genome sequencing can extract this additional layer of detail from the same sequencing budget, rather than adopting a new platform.

Three published methods layer a dsDNA deaminase onto Tn5-based ATAC-seq. Interestingly, each employs a different order of operations. For example, FOODIE⁴ generates high-resolution accessibility and TF footprinting maps by first tagmenting DNA with Tn5 followed by deamination using DddB or MGYPDa829. ACCESS-ATAC⁵ either concurrently or sequentially treats nuclei with deaminase (DddA or DddSs, aka SsDddA) and then Tn5. The authors concluded that concurrent enzyme addition yielded the most optimal results.  Lastly, cFOOT-seq⁶ pairs a deaminase, SsdAtox, with whole-genome sequencing rather than a targeted panel, extending TF footprinting genome-wide. This approach is also compatible with existing ATAC-seq and scATAC-seq pipelines. When combining deamination with tagmentation, the group found distinct advantages depending on whether deamination came before or after Tn5 tagmentation, suggesting the optimal ordering is dependent on the overall experimental goals and the specific research question being asked. Together, these examples show that while there’s not yet consensus on a single best way to pair a deaminase with Tn5 tagmentation, each approach lets researchers extract more precise protein footprinting information from the short-read workflows they already run.

Concluding Remarks

Overall, deaminase-based mapping methods achieve superior footprinting resolution compared to traditional cleavage-based approaches alone. Because deamination marks DNA rather than cutting it, and produces a change that survives PCR amplification, deaminase-coupled workflows can work from lower-input samples than methylation-based methods. Deaminase-based mapping methods can also resolve co-binding of multiple proteins along the same DNA molecule at single-molecule resolution when coupled with long-read sequencing.

Adoption of these exciting new technologies depends upon broad deaminase availability. Until recently, few dsDNA deaminases had been characterized, and none were commercially available, so researchers had to express and purify the enzyme in-house before they could even begin optimizing an assay. That is changing. NEB recently published a broad panel of dsDNA deaminases, including methylation-sensitive variants. EpiCypher has made SsDddA and its inhibitor SsDddI commercially available as purified, activity-confirmed exploratory reagents in the IDEA Toolbox. These resources expand the innovation possible around deaminase-based methods and lower the barrier for labs that want to adopt or build on the assays described above.

As more of these enzymes become available beyond specialized labs, it’s reasonable to expect continued refinement of existing deaminase-based assays as well as new applications, thereby extending deamination-based footprinting to additional biological contexts. The methods are still young, but they already begin to address a question genomics has struggled to answer directly: not only where a variant or a perturbation sits in the genome, but what it does to the surrounding chromatin on the same DNA molecule.

By: Emily A. Madden and Bryan J. Venter