Structure-activity relationships study on inhibition of CRISPR-Cas9 by glycosaminoglycans.

The CRISPR-Cas9 system is a revolutionary genome editing system known for its precision, simplicity and efficiency, playing a crucial role in gene-editing. It has enabled applications ranging from biomedicine to agriculture. However, the uncontrollable activity of Cas9 has raised significant safety concerns in clinical settings, limiting its broader application. Consequently, regulating CRISPR-Cas9 activity holds substantial promise for enhancing the safety and efficacy of gene-editing technologies. In this study, we demonstrated that sulfated glycosaminoglycans (GAGs) exhibit inhibitory effects of Cas9. Specifically, both chondroitin sulfate (CS) and heparin (HP) can inhibit CRISPR/Cas9 activity, with heparin showing a stronger inhibitory effect that correlates positively with its concentration. Molecular dynamics simulations indicate that sulfated heparin residues might inhibit Cas9 function by binding to essential DNA-binding sites, which are crucial for functional interactions, potentially impairing activity. Additionally, higher molecular weight GAGs exhibit enhanced inhibitory effects under the same sulphation. Notably, the sulfation site also influenced activity. C6-sulfation of chondroitin sulfate is more favorable for Cas9 inhibition, and N-sulfation of heparin enhances its inhibitory effect on Cas9 activity. These findings provide valuable insights into the development of carbohydrate-based inhibitors for CRISPR-Cas9, offering a foundation for further exploration in this field.