Chemical control of 2'-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement.

ABSTRACT: Advanced CRISPR-based therapies benefit from CRISPR RNA (crRNA) with high nuclease resistance and enhanced drug-like properties, which is primarily achieved through chemical replacement of the RNA ribose moiety. However, for gene editing enzymes like CRISPR-Cas9 a handful of residues cannot be replaced with chemical ribose analogues, limiting the scope of therapeutic strategies. The mechanism underlying this restriction has remained unclear. Here, using nucleic acid chemistry, biochemistry, cryo-EM, and molecular dynamics simulations, we show that the ribose 2'-hydroxyl group at specific crRNA residues is required to achieve a conformational state competent for Cas9 target DNA binding. Based on the mechanistic principles uncovered, we combined site-specific phosphorothioate linkage chemist

SUBMITTER: Pater AA 

PROVIDER: S-EPMC12874002 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature