While we have seen many contenders for the title of heavyweight champion in the epigenetic editing ring, the adage “big things come in small packages” has taken on a new meaning. Nanobodies, or single-domain antibodies, demonstrate that featherweights can pack an explosive epigenetic editing punch by using their opponents’ momentum against them.
Researchers led by Lacramioara Bintu (Stanford University, Stanford, California, USA) knew that employing components of chromatin regulators as effector domains for epigenetic editing entails a massive problem. Their large size makes it challenging to cram them into a single vector for delivery into mammalian cells. As a small-sized solution, the team looked to explore nanobodies as a minuscule means to recruit the cells’ own chromatin regulators in a hugely interesting innovative approach to the epigenetic control of gene expression.
To find their champion, the team tested out a few gene-silencing systems, which involved small expression plasmids coding for the reverse tetracycline repressor (rTetR) DNA-binding domain fused to either an anti-DNMT1 nanobody, an anti-HP1 nanobody, or the well-described KRAB repressor domain. The punching bag was a reporter gene within the AAVS1 “safe harbor” locus in human embryonic kidney cells, where doxycycline induction prompts rTetR binding to Tet operator-binding sites upstream of the reporter gene.
Here’s what they found after hitting the gym:
- rTetR-anti-HP1 and rTetR-anti-DNMT1 induce slow epigenetic silencing of reporter gene expression, although to a significantly lower degree than rTetR-KRAB
- While rTetR-anti-HP1-mediated silencing reverses after doxycycline withdrawal, rTetR-KRAB and rTetR-anti-DNMT1 pack a long-lasting punch and mediate long-term silencing to impart epigenetic memory
The team next explored advanced plasmid-based training strategies using their champion – the anti-DNMT1 nanobody – which involved evaluating several styles in the qualifying round:
- rTetR-KRAB-anti-DNMT1 allows robust gene silencing and extended epigenetic memory
- By removing the reliance on rTetr and enabling CRISPR-based targeting, dCas9-KRAB-anti-DNMT1 combined with specific guide RNAs mediates robust silencing and extends epigenetic memory at the reporter gene (but not a selected endogenous gene)
With nanobody-mediated recruitment of chromatin regulators entering the ring, the authors have turned one small package into a giant leap towards new epigenetic editing frontiers. Additionally, the team expects that tools such as rTetR-anti-DNMT1 will find use as “devices” in synthetic circuits as cellular stopwatches and recording devices.
Step into the epigenetic editing ring with this new contender over at Nature Communications, January 2021.