Application (Liu et al., 2016): Compare the mechanistic goals of fusing dCas9 to Tet1 versus dCas9 to Dnmt3a. Why is using a catalytically inactive Cas9 crucial for these experiments?

What is the mechanistic goal of fusing dCas9 to Tet1?

To erase DNA methylation at specific loci via active demethylation, activating gene expression.

What is the mechanistic goal of fusing dCas9 to Dnmt3a?

To establish de novo DNA methylation at unmethylated sites, silencing gene expression.

How do the goals of dCas9-Tet1 and dCas9-Dnmt3a compare?

They are opposite sides of targeted epigenetic editing: Tet1 activates genes by erasing methylation; Dnmt3a silences genes by adding methylation.

What is Tet1?

A dioxygenase that initiates active demethylation.

What happens when dCas9-Tet1 is targeted to a methylated and silenced promoter (e.g., Snrpn or BDNF)?

It removes inhibitory methyl marks, thereby activating gene expression.

What is Dnmt3a?

A de novo DNA methyltransferase.

What happens when dCas9-Dnmt3a is targeted to an active promoter (e.g., Gapdh reporter)?

It adds methyl marks to silence gene expression.

What additional use does dCas9-Dnmt3a have beyond silencing genes?

It can methylate CTCF binding sites, which blocks CTCF recruitment and disrupts DNA loops.

Why is using catalytically inactive Cas9 (dCas9) crucial for epigenetic editing?

It allows editing of the epigenetic state without altering the underlying DNA sequence, ensuring observed changes are causally linked to methylation changes rather than genetic damage.

What would happen if active Cas9 were used instead of dCas9?

Active Cas9 would create double-strand breaks and introduce genetic mutations at the target site, confounding interpretation of changes in gene expression or cell behavio