Molecular basis for methylation-sensitive editing by Cas9 - PubMed
2 hours ago
- #genome editing
- #DNA methylation
- #CRISPR-Cas9
- ThermoCas9 is a methylation-sensitive Cas9 variant that binds and cleaves DNA with specific PAM sequences (5'-NNNNCGA-3' or 5'-NNNNCCA-3') in vitro, but its activity is inhibited when the fifth cytosine in these PAMs is methylated (5mCpG or 5mCpC).
- Cryo-EM structures at 2.8 Å and 2.2 Å resolution reveal the molecular basis for ThermoCas9's requirement of an unmethylated cytosine in the PAM, providing insights for enzyme engineering.
- ThermoCas9 demonstrates methylation-sensitive editing in human cell lines with distinct DNA methylation landscapes and can target hypomethylated genes, such as luminal expression signature genes in breast cancer, adding precision to genome-editing technologies.
- A patent application related to methylation-sensitive gene editing using ThermoCas9 has been filed, with multiple institutions and inventors involved, and one author discloses advisory roles in biotech companies.