ThermoCas9: a methylation-sensitive CRISPR enzyme enabling epigenetic-aware editing
ThermoCas9 is a thermostable Cas9 whose activity is blocked by methylation of the PAM cytosine (5mC) in PAMs 5′-NNNNCGA-3′ or 5′-NNNNCCA-3′, restricting binding and cleavage to unmethylated sites. Cryo-EM structures of pre- and post-cleavage states reveal how PAM bases are recognized and why methylation prevents engagement. In human cells, unmethylated PAMs edit efficiently whereas methylated ones do not; a catalytically enhanced ThermoCas9 and optimized delivery methods boost editing at hypomethylated breast cancer loci ESR1 and GATA3, illustrating a new layer of precision for genome engineering and potential epigenetic screening applications.