Nucleosome positioning on DNA governs gene accessibility by controlling the binding of transcription factors and RNA polymerases. Recent studies have suggested that nucleosomes can directly inhibit the activity of the CRISPR-Cas9 system. Despite promising prospects for therapeutic applications, the molecular details of nucleosome-mediated CRISPR inhibition are not well understood. Researchers from the University of California, Berkeley, and the University of California, San Francisco have now shown that nucleosomes inhibit CRISPR-Cas9 activity by disrupting the formation of the R-loop intermediate.

Cas9 is a nuclease responsible for introducing DNA double-strand breaks at target sites specified by a synthetic guide RNA. To initiate DNA cleavage, Cas9 must first form a stable complex with the target DNA, which involves unwinding the DNA duplex to form an R-loop structure. The researchers found that nucleosomes can interfere with this process by preventing the formation of the R-loop intermediate. This inhibition is dependent on the positioning of the nucleosome relative to the target site and can be relieved by histone modifications or by altering the DNA sequence.

These findings provide insights into the molecular mechanisms of CRISPR-Cas9 inhibition by nucleosomes and suggest strategies to improve the efficiency and specificity of CRISPR-based gene editing technologies.