Targeted bisulfite sequencing after chromatin effector recruitment in HEK293T cells

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, consisting of multiple domains that are necessary for their function, either to stimulate catalytic function or to mediate interactions with other members of the complexes they are part of. In addition, the most efficient way to control gene expression and impart epigenetic memory is to engage multiple chromatin regulators in a combinatorial fashion. Due to the large size of chromatin regulators and the requirement to use them in combinations, it is difficult to build compact gene regulatory tools that can fit in viral delivery vectors such as AAV or lentivirus. To overcome these current issues with epigenome editing tools, we reasoned that we could build a smaller tool for gene regulation that could easily be combined and delivered by using small (~15 kDa) single-domain antibodies (also called nanobodies) to recruit endogenous chromatin regulators from the existing cellular chromatin network. We show that targeted recruitment of antiDNMT1, a nanobody that binds to the DNA methyltransferase 1, to a TagRFP reporter gene located at the AAVS1 safe harbor site in HEK293T cells leads to gene silencing and DNA methylation at this site (as shown by targeted bisulfite sequencing). Moreover, the DNA methylation levels observed with antiDNMT1 were comparable to direct DNMT1 recruitment but were lower than those obtained after recruitment of DNMT3B.