Bringing in the right combination of people with different perspectives and abilities can take a team to new heights. And just like that savvy diverse team, a new epigenome editing tool called the Spy-Snoop-Sun-Avi (SSSavi) system recruits diverse effector molecules in an interchangeable and orderly way to understand how combinations of DNA and histone modifications affect gene expression.
Currently, crafty researchers can edit the epigenome by directly fusing a DNA binding domain—often a deactivated Cas9 (dCas9)—to an effector molecule, such as DNMT3A for targeted methylation. But such constructs haven’t always worked as expected because in real-world contexts, some effectors need to bind additional factors or have other effectors nearby.
To address this shortcoming, Ryan Lister’s savvy group (Harry Perkins Institute of Medical Research) previously turned to the SunTag system, which can bind multiple effectors on a repetitive GCN4 peptide array fused to dCas9. But they still couldn’t control the stoichiometry or spatial order of the effector team because they all had to be bound to the same antibody to dock.
Building on this previous work, Swain et al. now report SSSavi, which can bind combinations of up to four different effectors in a precise, controllable spatial pattern. Here’s what they found in HEK293T cells:
- dCas9 is fused to the Spy, Snoop, Sun, and Avi tags, which are placed in series—one after the other
- Different effectors are bound to the tags’ respective catchers: SpyCatcher, SnoopCatcher, the GCN4 antibody, or Traptavidin
- The tags’ locations are interchangeable, so many spatial combinations are possible
- In tests with a transcriptional activator, activation levels were generally similar whether tags or catchers were fused to dCas9, or whether the tags or catchers were fused to the activaton
- In a head-to-head comparison, SSSavi generally performed similarly to the SunTag system and to an effector fused directly to dCas9
The team then bound three repressive effectors, DNMT3A, KRAB, and EZH2 to the four catchers and found some differences, depending on the gene being repressed and the catcher used. So, the savvy experimenters combined effectors in various ways to see if they could improve repression. Here’s what they found:
- Combining some of the effectors in the SSSavi system increased repression
- The spatial arrangements of the effectors impacted repression at some genes
- For three of the four genes tested, the strongest effect was seen when the tags placed the molecules in this order: DNMT3A-KRAB-EZH2
- Some combinations were synergistic, meaning the effects were greater than the sum of the individual effects
When cells were transfected with the DNMT3A-KRAB-EZH2 SSSavi system, the researchers found increased DNA methylation at all six tested genes, as well as a reduction in H3K4me3, an activating modification. They didn’t see changes, however, in H3K9me3 or H3K27me3 levels.
And when the team tested the system in a clinically relevant liver cancer cell line, they found that SSSavi with the DNMT3A-KRAB-EZH2 effector order significantly downregulated EPCAM, a biomarker of hepatocellular carcinoma.
Overall, SSSavi is a very savvy, versatile epigenomic editing tool that shrewd researchers can arrange as they wish to test the spatial effects and combinations of many different effectors simultaneously.
To be well-informed and knowledgeable (i.e., savvy) about SSSavi, head over to Nucleic Acids Research, November 2023.